Fusion molecules and IL-15 variants

ABSTRACT

The instant invention provides soluble fusion protein complexes and IL-15 variants that have therapeutic and diagnostic use, and methods for making the such proteins. The instant invention additionally provides methods of stimulating or suppressing immune responses in a mammal using the fusion protein complexes and IL-15 variants of the invention.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/928,900, filed May 11, 2007, the entire contents of which arehereby incorporated herein by reference.

GOVERNMENT SUPPORT

Research supporting this application was carried out by the UnitedStates of America as represented by the Secretary, Department of Healthand Human Services.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 26, 2010, isnamed 68295.txt and is 54,760 bytes in size.

BACKGROUND OF THE INVENTION

T Cell Receptors (TCR) are primary effectors of the immune system thathave unique advantages as a platform for developing therapeutics. Whileantibody therapeutics are limited to recognition of pathogens in theblood and extracellular spaces or to protein targets on the cellsurface, T cell receptors can recognize antigens displayed with MHCmolecules on the surfaces of cells (including antigens derived fromintracellular proteins). Depending on the subtype of T cells thatrecognize displayed antigen and become activated, T cell receptors and Tcells harboring T cell receptors can participate in controlling variousimmune responses. For instance, T cells are involved in regulation ofthe humoral immune response through induction of differentiation of Bcells into antibody producing cells. In addition, activated T cells actto initiate cell-mediated immune responses. Thus, T cell receptors canrecognize additional targets not available to antibodies.

T-cells are a subgroup of cells which together with other immune celltypes (polymorphonuclear, eosinophils, basophils, mast cells, B—, NKcells) constitute the cellular component of the immune system. Underphysiological conditions T-cells function in immune surveillance and inthe elimination of foreign antigen. However, under pathologicalconditions there is compelling evidence that T-cells play a major rolein the causation and propagation of disease. In these disorders,breakdown of T-cell immunological tolerance, either central orperipheral is a fundamental process in the causation of autoimmunedisease.

The TCR is believed to play an important role in the development andfunction of the immune system. For example, the TCR has been reported tomediate cell killing, increase B cell proliferation, and impact thedevelopment and severity of various disorders including cancer,allergies, viral infections and autoimmune disorders.

It thus would be desirable to provide novel targeting agents based on Tcell receptors, as well as methods for producing and using such agentsfor therapeutic and diagnostic settings. Accordingly, it would beparticularly desirable to provide such molecules that would have certainadvantages in comparison to prior art complexes based on antibodytargeting.

Moreover, it is desirable to use the TCR to target various effectormolecules to the disease site where they can provide therapeutic benefitwithout the side effects associated with system non-targeted activity.One such is IL-15, a member of the four alpha-helix bundle family oflymphokines. IL-15 plays a multifaceted role in development and controlof the immune system. More specifically, IL-15 influences the function,development, survival, and proliferation of CD8+ T cells, NK cells,killer T cells, B cells, intestinal intraepithelial lymphocytes (IEL)and antigen-presenting cells (APC). It has been demonstrated that bothIL-15−/−, and IL-15Ra−/− transgenic mice lack peripheral NK and killer Tcell populations, certain IEL subsets, and most memory phenotype CD8+ Tcells, suggesting IL-15 plays role in the development, proliferationor/and survival of these cell types. The IL-15 receptor (R) consists ofthree polypeptides, the type-specific IL-15R alpha (“IL-15Rα” or“IL-15Ra”), the IL-2/IL-15Rbeta (“IL-2Rβ” or “IL-15Rb”), and the commongamma chain (“γC,” or “gC” which is shared by multiple cytokinereceptors).

IL-15 signaling can occur through the heterotrimeric complex of IL-15Rα,IL-2Rβ and γC; through the heterodimeric complex of IL-2Rβ and γC. Anovel mechanism of IL-15 action is that of transpresentation in whichIL-15 and IL-15Rα are coordinately expressed by antigen-presenting cells(monocytes and dendritic cells), and IL-15 bound to IL-15Rα is presentedin trans to neighboring NK or CD8 T cells expressing only the IL-15RβγCreceptor. As a co-stimulatory event occurring at the immunologicalsynapse, IL-15 transpresentation now appears to be a dominant mechanismfor IL-15 action in vivo and appears to play a major role in tumorimmunosurveillance (Waldmann, T A, 2006, Nature Rev. Immunol.6:595-601). Soluble IL-2Rα subunits, inducing isoforms containing adeletion of exon3 and the so-called “sushi” domain at the N terminus,have been shown to bear most of the structural elements responsible forcytokine binding. Whereas IL-2Rα alone is a low affinity receptor forIL-2 (Kd_(—)10 nM), IL-15Rα binds IL-15 with high affinity (Kd_(—)100pM). Thus soluble IL-2Rα and IL-15 are able to form stable heterodimericcomplexes in solution and these complexes are capable of modulating(i.e. either stimulating or blocking) immune responses via theintermediate or high affinity IL-15R complex (Mortier et al. 2006. JBiol Chem 281: 1612-1619; Stoklasek et al. 2006. J Immunol 177:6072-6080; Rubinstein et al. 2006. Proc Natl Acad Sci USA 103:9166-9171).

Given the known effects of IL-15 on the immune system, a number ofIL-15-based approaches have been explored to manipulate the immunesystem for the hosts benefit. While IL-15 administration has beenemployed to bolster immune responses or augment immune systemreconstitution, blockade of IL-15 activity can inhibit autoimmune andother undesirable immune responses (Waldmann, T A, 2006, Nature Rev.Immunol. 6:595-601). In fact, one of the limitations with systemic IL-15treatment is the possible induction of autoimmune disease. Otherlimitations include the difficulty in produce this cytokine in standardmammalian cell expression systems as well as its very short half-life invivo. Therefore, there is a need to generate a suitableimmunostimulatory therapeutic form of IL-15 that displays a longer invivo half-life, increased activity binding to immune cells, or enhancedbioactivity. Additionally there is a need for effective IL-15antagonists. Ideally it would be desirable that such molecules beselectively targeted to the disease site to avoid unwanted systemictoxicities and provide a more effective therapeutic benefit.

SUMMARY OF THE INVENTION

The instant invention provides a number of IL-15 variants and solublefusion complexes that have therapeutic use and methods for making suchproteins. The instant invention provides methods for killing targetcells using the soluble fusion complexes of the invention. The IL-15variants and soluble complexes described herein have potentialtherapeutic utility.

Accordingly, in one aspect, the invention provides a soluble fusionprotein complex comprising at least two soluble fusion proteins, whereinthe first fusion protein comprises a first biologically activepolypeptide covalently linked to interleukin-15 (IL-15) or functionalfragment thereof, and the second fusion protein comprises a secondbiologically active polypeptide covalently linked to solubleinterleukin-15 receptor alpha (IL-15Ra) polypeptide or functionalfragment thereof, wherein IL-15 domain of a first fusion protein bindsto the soluble IL-15Ra domain of the second fusion protein to form asoluble fusion protein complex.

In one embodiment, one of the first and second biologically activepolypeptides comprises a first soluble T-cell receptor (TCR) orfunctional fragment thereof. In another embodiment, an other of thebiologically active polypeptides comprises the first soluble TCR orfunctional fragment thereof, thereby creating a multivalent TCR fusionprotein complex with increased binding activity. In a furtherembodiment, the other biologically active polypeptide comprises a secondsoluble TCR or functional fragment thereof, different than the firstsoluble TCR.

In another embodiment of the aspect, the TCR is specific for recognitionof a particular antigen.

In a further embodiment of the aspect, the TCR is a heterodimercomprising a and b chain TCR.

In still another embodiment of the aspect, the TCR comprises a singlechain TCR polypeptide. In a further embodiment, the single chain TCRcomprises a TCR V-α chain covalently linked to a TCR V-β chain by apeptide linker sequence. In another further embodiment, the single chainTCR further comprises a soluble TCR Cβ chain fragment covalently linkedto a TCR V-β chain.

In another embodiment, the single chain TCR further comprises a solubleTCR Cα chain fragment covalently linked to a TCR V-α chain.

In a further embodiment, one or both of the first and secondbiologically active polypeptides comprises an antibody or functionalfragment thereof.

In still another embodiment, the antibody is specific for recognition ofa particular antigen. In a further embodiment, the antibody is asingle-chain antibody or single-chain Fv. In another particularembodiment, the single-chain antibody comprises an immunoglobulin lightchain variable domain covalently linked to immunoglobulin heavy chainvariable domain by polypeptide linker sequence.

In one embodiment of the above described aspects, the first biologicallyactive polypeptide is covalently linked to IL-15 (or functional fragmentthereof) by polypeptide linker sequence.

In another embodiment of the above described aspects, the secondbiologically active polypeptide is covalently linked to IL-15Rapolypeptide (or functional fragment thereof) by polypeptide linkersequence.

In another embodiment, the antigen for the TCR domain comprises peptideantigen presented in an MHC or HLA molecule. In a further embodiment,the peptide antigen is derived from a tumor associated polypeptide orvirus encoded polypeptide.

In another embodiment, the antigen for the antibody domain comprises acell surface receptor or ligand.

In a further embodiment, the antigen comprises a CD antigen, cytokine orchemokine receptor or ligand, growth factor receptor or ligand, tissuefactor, cell adhesion molecule, MHC/MHC-like molecules, FC receptor,Toll-like receptor, NK receptor, TCR, BCR, positive/negativeco-stimulatory receptor or ligand, death receptor or ligand, tumorassociated antigen, or virus encoded antigen.

In another embodiment of the above described aspects, the IL-15Rapolypeptide comprises the extracellular domain of the IL-15 receptoralpha capable for binding IL-15.

In another embodiment of the above described aspects, the IL-15Rapolypeptide comprise either the IL-15a sushi domain (Wei et al. Journalof Immunology, 2001, 167: 277-282) or the IL-15aΔE3 domain (Anderson etal. 1995. J. Biol. Chem. 270:29862-29869, Dubois et al. 1999. J. Biol.Chem. 274:26978-26984).

In another aspect, the invention provides for an IL-15 variant (alsoreferred to herein as IL-15 mutant) that has a different amino acidsequence that the native (or wild type) IL-15 protein and that binds theIL-15Ra polypeptide and functions as an IL-15 agonist or antagonist.Embodiments of the invention provide an IL-15 variant as a non-fusionprotein or as a soluble fusion protein comprising a biologically activepolypeptide described above, wherein the IL-15 variant is used in placeof the IL-15 domain.

In one embodiment of the above described aspects, the invention featuresa nucleic acid sequence encoding the first fusion protein of any of theaspects or embodiments as described herein.

In one embodiment of the above described aspects, the invention featuresa nucleic acid sequence encoding the second fusion protein of any of theaspects or embodiments as described herein.

In one embodiment of the above described aspects, the invention featuresa nucleic acid sequence encoding the IL-15 variant of any of the aspectsor embodiments as described herein.

In a one embodiment, the nucleic acid sequence further comprises apromoter, translation initiation signal, and leader sequence operablylinked to the sequence encoding the fusion protein or IL-15 variant. Inanother embodiment, any of the nucleic acid sequences as described aboveare contained in a DNA vector.

In another aspect, the invention features a method for making a solublefusion protein complex of the above-described aspects, the methodcomprising introducing into a first host cell a DNA vector of theabove-described aspects and embodiments that encodes the first fusionprotein, culturing the first host cell in media under conditionssufficient to express the first fusion protein in the cell or the media,purifying the first fusion protein from the host cells or media,introducing into a second host cell a DNA vector of the above-describedaspects and embodiments encoding the second fusion protein, culturingthe second host cell in media under conditions sufficient to express thesecond fusion protein in the cell or the media, and purifying the secondfusion protein from the host cells or media, and mixing the first andsecond fusion protein under conditions sufficient to allow bindingbetween IL-15 domain of a first fusion protein and the soluble IL-15Radomain of a second fusion protein to form the soluble fusion proteincomplex.

In another aspect, the invention features a method for making a solublefusion protein complex of the above-described aspects, the methodcomprising introducing into a host cell a DNA vector of theabove-described aspects and embodiments, encoding the first fusionprotein and a DNA vector as described in the above-described aspects andembodiments, encoding the second fusion protein, culturing the host cellin media under conditions sufficient to express the fusion proteins inthe cell or the media and allow association between IL-15 domain of afirst fusion protein and the soluble IL-15Ra domain of a second fusionprotein to form the soluble fusion protein complex, purifying thesoluble fusion protein complex from the host cells or media.

In still another aspect, the invention features a method for making asoluble fusion protein complex of the above-described aspects, themethod comprising introducing into a host cell a DNA vector of claim 28encoding the first and second fusion proteins, culturing the host cellin media under conditions sufficient to express the fusion proteins inthe cell or the media and allow association between IL-15 domain of afirst fusion protein and the soluble IL-15Ra domain of a second fusionprotein to form the soluble fusion protein complex, purifying thesoluble fusion protein complex from the host cells or media.

In still other aspects of the above described methods, the DNA vectorencoding the IL-15 variant is used in place of the DNA vector encodingthe first fusion protein to generate a host cell capable of expressingthe IL-15 variant and the IL-15 variant is allowed associate with theIL-15Ra domain of a second fusion protein to form a soluble fusionprotein complex.

In another aspect, the invention features a method for making an IL-15variant of the above-described aspects, the method comprisingintroducing into a host cell a DNA vector of the above-described aspectsand embodiments that encodes an IL-15 variant, culturing the host cellin media under conditions sufficient to express the IL-15 variant in thecell or the media, purifying the an IL-15 variant from the host cells ormedia.

In another aspect, the invention features a method for killing a targetcell, the method comprising contacting a plurality of cells with asoluble fusion protein complex or IL-15 variant of any of theabove-described aspects or embodiments, wherein the plurality of cellsfurther comprises immune cells bearing the IL-15R chains recognized bythe IL-15 domain of the above-described aspects and the target cellsbearing an antigen recognized by at least one of the biologically activepolypeptides of the above-described aspects, forming a specific bindingcomplex (bridge) between the antigen on the target cells and the IL-15Rchains on the immune cells sufficient to bind and activate the immunecells, and killing the target cells by the bound activated immune cells.

In one embodiment of the method, the target cells are tumor cells orvirally infected cells.

In another embodiment of the method, the biologically active polypeptidecomprises a TCR.

In still another embodiment of the method, the antigen on the targetcells comprises a tumor or virally encoded peptide antigen presented inan MHC or HLA molecule and recognized by the TCR.

In a further embodiment of the method, the immune cells are T-cells, LAKcells or NK cells.

In another aspect, the invention features a method for preventing ortreating disease in a patient in which the diseased cells express adisease associated antigen, the method comprising administering to thepatient a soluble fusion protein complex or IL-15 variant of any of theabove-described aspects or embodiments, comprising a biologically activepolypeptide recognizing a disease-associated antigen forming a specificbinding complex (bridge) between antigen-expressing diseased cells andIL-15R-expressing immune cells sufficient to localize the immune cells,and damaging or killing the disease cells sufficient to prevent or treatthe disease in the patient.

In one aspect, the invention features a method for preventing ortreating disease in a patient in which the diseased cells express adisease associated antigen, the method comprising mixing immune cellsbearing the IL-15R chains with a soluble fusion protein complex of claim1-22 comprising a biologically active polypeptide recognizing adisease-associated antigen, administering to the patient the immunecell-fusion protein complex mixture, forming a specific binding complex(bridge) between antigen-expressing diseased cells and IL-15R-expressingimmune cells sufficient to localize the immune cells; and damaging orkilling the disease cells sufficient to prevent or treat the disease inthe patient.

In another aspect, the invention features a method for preventing ortreating an disease in a patient in which the patient's cells express adisease associated antigen, the method comprising administering to thepatient a soluble fusion protein complex or IL-15 variant of any of theabove-described aspects or embodiments, comprising a biologically activepolypeptide recognizing a disease-associated antigen on the patient'scells, localizing soluble fusion protein complex or IL-15 variant on thepatient's cells wherein the IL-15 domain of the soluble fusion proteincomplex or IL-15 variant binds immune cells bearing the IL-15R chainsand suppressing the immune response of the immune cells.

In one embodiment of the method, the disease is cancer or viralinfection.

In another embodiment of the method, the disease is an immune disorder,autoimmune disease or inflammatory disorder.

In another embodiment of the method, the disease associated antigen is apeptide/MHC complex.

In another embodiment, the invention features a method of stimulatingimmune responses in a mammal comprising administering to the mammal aneffective amount of the soluble fusion protein complex or IL-15 variantof any of the above-described aspects and embodiments.

In another embodiment, the invention features a method of suppressingimmune responses in a mammal comprising administering to the mammal aneffective amount of the soluble fusion protein complex or IL-15 variantof any of the above-described aspects and embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1(A & B) are schematic drawings. (A) is a schematic depicting anexample of a fusion protein complex containing single chain TCRpolypeptides. (B) is a schematic depicting representative fusion proteinconstructs comprising the fusion protein complex (SEQ ID NO: 3).

FIG. 2 (A-C) consists of three panels. (A) depicts a map ofpNEF38-c264scTCR/huIL15 expression vector. (B) shows the sequence ofc264scTCR/huIL15 fusion gene (SEQ ID NO: 4) and (C) shows the sequenceof c264scTCR/huIL15 fusion protein (SEQ ID NO: 5), including the leadersequence.

FIG. 3 (A-C) consists of three panels. (A) depicts a map ofpNEF38-c264scTCR-hinge-huIL15 expression vector. (B) shows the sequenceof c264scTCR-hinge-huIL15 fusion gene (SEQ ID NO: 6) and (C) shows thesequence of c264scTCR-hinge-huIL15 fusion protein (SEQ ID NO: 7,including the leader sequence.

FIGS. 4 (A-C) consists of three panels. (A) depicts a map ofpNEF38-c264scTCR/huIL15RaDE3 expression vector. (B) shows the sequenceof c264scTCR/huIL15RαΔE3 fusion gene (SEQ ID NO: 8) and (C) shows thesequence of c264scTCR/huIL15RαΔE3 fusion protein (SEQ ID NO: 9),including the leader sequence.

FIG. 5 (A-C) consists of three panels. (A) depicts a map of thepNEF38-c264scTCR/huIL15RaSushi expression vector. (B) shows the sequenceof c264scTCR/huIL15RαSushi fusion gene (SEQ ID NO: 10) and (C) shows thesequence of c264scTCR/huIL15RαSushi fusion protein (SEQ ID NO: 11),including the leader sequence.

FIG. 6 (A-C) consists of three panels. (A) depicts thepNEF38-c264scTCR-hinge-huIL15RaSushi expression vector. (B) shows thesequence of c264scTCR-hinge-huIL15RαSushi fusion gene (SEQ ID NO: 12)and (C) shows the sequence of c264scTCR-hinge-huIL15RαSushi fusionprotein (SEQ ID NO: 13), including the leader sequence.

FIG. 7 is a map of pSun-c264scTCRIL15/c264scTCRIL15RaSushi expressionvector.

FIG. 8 is a map of pSun-c264scTCRIL15/c264scTCRIL15RaDE3 expressionvector.

FIGS. 9(A & B) set forth characterization of transfected cellsexpressing TCR/IL15Rα fusion protein. (A) is two graphs showing flowcytometric analysis of fusion protein expressing cells. (B) is a graphshowing TCR-based ELISA results for fusion protein production.

FIGS. 10(A & B) shows analysis of TCR/IL15 and TCR/IL15Rα fusionproteins by reducing SDS PAGE. (A) shows cell culture supernatantscontaining c264scTCR/huIL15 or c264scTCR/huIL15RaSushi. (B) shows cellculture supernatants containing c264scTCR/huIL15 orc264scTCR/huIL15RαΔE3.

FIGS. 11(A-C) shows analysis of TCR/IL15, TCR/IL15Ra and fusion proteincomplexes by size exclusion chromatography. (A) is a graph showing theSEC chromatography profile of c264scTCR/huIL15. (B) is a graph showingthe SEC chromatography profile of c264scTCR/huIL15RαSushi. (C) is agraph showing the SEC chromatography profile ofc264scTCR/huIL15+c264scTCR/huIL15RαSushi fusion protein complex.

FIGS. 12(A & B) is an analysis of TCR/IL15Rα and fusion proteincomplexes by size exclusion chromatography. (A) is a graph illustratingthe SEC chromatography profile of c264scTCR/huIL15RαΔE3. (B) is a graphillustrating the SEC chromatography profile ofc264scTCR/huIL15+c264scTCR/huIL15RαΔE3 fusion protein complex.

FIG. 13 is a graph showing the binding of TCR/IL15, TCR/IL15Rα andfusion protein complexes to peptide/MHC complexes displayed on cells, asdetermined by flow cytometry.

FIG. 14 (A-D) consists of four panels. (A) shows the sequence of maturehuman IL15 protein (SEQ ID NO:1) and the blue underlined residues aresubstituted in the IL-15 variants as showed in Table 1. (B) depicts thepNEF38-c264scTCR-hinge-huIL15D8A and pNEF38-c264scTCR-hinge-huIL15D8Nexpression vectors. (C) shows the sequence ofpNEF38-c264scTCR-hinge-huIL15D8A (SEQ ID NO: 14) andpNEF38-c264scTCR-hinge-huIL15D8N (SEQ ID NO: 15) genes and (D) shows thesequence of pNEF38-c264scTCR-hinge-huIL15D8A (SEQ ID NO: 16) andpNEF38-c264scTCR-hinge-huIL15D8N fusion protein (SEQ ID NO: 17),including the leader sequence. The blue underlined nucleotides werechanged to generate the indicated IL-15 variants.

FIG. 15 is a graph showing flow cytometric analysis of IL-15R-bearingCTLL2 cells stained with fusion proteins and complexes following byTCR-specific peptide/MHC reagent.

FIG. 16 depicts CTTL-2 cell proliferation assays of IL15 activity fordimeric fusion proteins complexes of TCR/IL15RαSushi and TCR/IL15,comprising native and variant forms of IL15, to cognate peptide/MHCcomplexes displayed on cells loaded with peptide, as determined by flowcytometry.

FIG. 17 (A-C) are graphs showing the binding of dimeric fusion proteinscomplexes of TCR/IL15RαSushi and TCR/IL15, comprising native and variantforms of IL15, to cognate peptide/MHC complexes displayed on cellsloaded with peptide, as determined by flow cytometry. Background bindingof the dimeric fusion proteins complexes on cells with no loaded peptideis also shown (A) is a graph showing the binding of the dimericcomplexes of TCR/IL15RαSushi and TCR/IL15 wt (native form), orTCR/IL15D8N or TCR/IL15D8A variants to cognate peptide/MHC complexesdisplayed on cells. (B) is a graph showing the slight background bindingof dimeric complexes of TCR/IL15RαSushi and TCR/IL15 wt (native form) tothe cells without loaded peptide. No background binding of dimericcomplexes of TCR/IL15RαSushi and TCR/IL15D8N or TCR/IL15D8A variants wasseen to the cells with not loaded. (C) is graph(showing flow cytometricanalysis of IL-15RβγC-bearing 32Dβ cells stained with dimeric complexesof TCR/IL15RαSushi and TCR/IL15 wt (native form), or TCR/IL15N72D,TCR/IL15D8N or TCR/IL15D8A variants. Enhanced IL-15RβγC binding of thecomplex containing TCR/IL15N72D and decreased IL-15RβγC binding ofcomplexes containing TCR/IL15D8N or TCR/IL15D8A was observed.

FIG. 18 (A&B) are graphs showing binding activities of wide type,antagonist, and agonist TCR/IL15 fusion proteins to cognate peptide/MHCcomplexes and IL15 Rα as determined by ELISA. (A) is analysis showingbinding activity of fusion proteins to cognate peptide/MHC complexes.(B) is analysis showing binding activity of fusion proteins to IL15Rα.

FIG. 19(A-C) are graphs showing the ability of TCR/IL-15 fusion proteinscomprising IL-15 variants to inhibit or enhance growth of IL15R-bearingcells, as determined by cell proliferation assay. (A) is graph showingthe activity of fusion proteins comprising IL-15 variants to inhibit theproliferation of high affinity IL15R (αβγ receptor complex) bearingCTLL-2 cells. (B) is graph showing the activity of fusion proteinscomprising IL-15 variants to inhibit or enhance the proliferation of lowaffinity IL15R (βγ receptor complex) bearing 32Dβ cells. (C) is graphshowing the activity of fusion proteins comprising IL-15 variants toblock TCR/IL15wt-stimulated proliferation of high affinity IL15R (αβγreceptor complex) bearing CTLL-2 cells.

FIG. 20 depicts the effects of in vitro incubation of NK cells withdimeric fusion proteins complexes of TCR/IL15RαSushi and TCR/IL15 on thesurvival of xenograft tumor-bearing nude mice. Athymic nude mice wereinjected with human NSCLC A549-A2 cells to allow establishment of lungmetastases. Purified NK cells isolated from spleens of allogenic donormice were incubated in vitro with rhIL-2, MART1scTCR-IL2, c264scTCR-IL2or c264scTCR-IL15/c264scTCR-IL15Rα and adoptively transferred into thetumor-bearing mice that had been pretreated with cyclophosphamide (CTX),as indicated in the figure legend. The percent survival followingtreatment was plotted.

FIG. 21 sets forth Table 1 showing the amino acid replacements in theIL-15 variants and the affects of these changes on IL-15 activity.

FIGS. 22A-B set forth the amino acid sequence of IL-15(SEQ ID NO:1) andthe nucleic acid sequence of IL-15 (SEQ ID NO:2), respectively.

DETAILED DESCRIPTION OF THE INVENTION

It has been established that the IL-15 stably binds to the extracellulardomain of the IL-15Rα and that the resulting complex is capable ofmodulating (i.e. either stimulating or blocking) immune responses viathe intermediate or high affinity IL-15R complex (Mortier, E. et al.,2006, J. Biol. Chem., 281: 1612-1619; Stoklasek, T. et al., 2006, JImmunol 177: 6072-6080; Rubinstein, M. P. et al., 2006, Proc Natl AcadSci U S A 103: 9166-9171, Waldmann, T. A., 2006, Nat Rev Immunol 6:595-601). In addition, it has been demonstrated that single-chain TCR orantibody polypeptides can be fused to cytokines and other immuneeffector domains and that such bispecific fusion molecules retainfunctional activity of both fusion domains (Belmont, H. J. et al., 2006,Clin Immunol 121: 29-39; Card, K. F. et al., 2004, Cancer ImmunolImmunother 53: 345-357; Mosquera, L. A. et al., 2005, J Immunol 174:4381-4388; Penichet, M. L., 1997, Hum Antibodies 8: 106-118). Further,it has been shown that multivalent forms of the TCR provide enhancedbinding to their ligands (Zhu, X., H. J., 2006, J Immunol 176:3223-3232).

Definitions

The following definitions are provided for specific terms which are usedin the following written description.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. The term “a nucleic acid molecule” includesa plurality of nucleic acid molecules.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude other elements. “Consisting essentially of”, when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants from the isolation and purification methodand pharmaceutically acceptable carriers, such as phosphate bufferedsaline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, chimeric and single-chain antibodies as well as bispecificantibodies.

The term “antigen” as used herein is meant any substance that causes theimmune system to produce antibodies or specific cell-mediated immuneresponses against it. A disease associated antigen is any substance thatis associated with any disease.

The term “biologically active polypeptide” as used herein is meant torefer to an amino acid sequence such as a protein, polypeptide orpeptide; a sugar or polysaccharide; a lipid or a glycolipid,glycoprotein, or lipoprotein that can produce the desired effects asdiscussed herein, including a TCR or antibody fusion protein complexwith antigen binding activity.

The term “cell” as used herein is meant to include any prokaryotic,eukaryotic, primary cell or immortalized cell line, any group of suchcells as in, a tissue or an organ. Preferably the cells are of mammalianand particularly of human origin, and can be infected by one or morepathogens. A “host cell” in accord with the invention can be atransfected, transformed, transduced or infected cell of any origin,including prokaryotic, eukaryotic, mammalian, avian, insect, plant orbacteria cells, or it can be a cell sof any origin that can be used topropagate a nucleic acid described herein.

The term “conjugate molecule” as it is used herein is meant to refer toa TCR or antibody molecule and an effector molecule usually a chemicalor synthesized molecule covalently linked (i.e. fused) by chemical orother suitable method. If desired, the conjugate molecule can be fusedat one or several sites through a peptide linker sequence or a carriermolecule. Alternatively, the peptide linker or carrier may be used toassist in construction of the conjugate molecule. Specifically preferredconjugate molecules are conjugate toxins or detectable labels.

The term “effector molecule” as used herein is meant to refer to anamino acid sequence such as a protein, polypeptide or peptide; a sugaror polysaccharide; a lipid or a glycolipid, glycoprotein, lipoprotein orchemical agent that can produce the desired effects as discussed herein,including an IL-15 domain, IL-15 variant or IL-15 receptor such asIL-15Rα, IL-2Rβ or γC, or functional fragments thereof.

The terms “fusion molecule” and “fusion protein” are usedinterchangeably and are meant to refer to a biologically activepolypeptide usually a TCR or antibody and an effector molecule usually aprotein or peptide sequence covalently linked (i.e. fused) byrecombinant, chemical or other suitable method. If desired, the fusionmolecule can be fused at one or several sites through a peptide linkersequence. Alternatively, the peptide linker may be used to assist inconstruction of the fusion molecule. Specifically preferred fusionmolecules are fusion proteins. Generally fusion molecule also can becomprised of conjugate molecules.

The term “host cell” is meant to refer to any prokaryotic or eukaryoticcell that contains either a cloning vector or an expression vector. Thisterm also includes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

The term “immune response” as used herein is meant to refer to theprocess whereby immune cells are stimulated and recruited from the bloodto lymphoid as well as non-lymphoid tissues via a multifactorial processthat involves distinct adhesive and activation steps. Activationconditions cause the release of cytokines, growth factors, chemokinesand other factors, upregulate expression of adhesion and otheractivation molecules on the immune cells, promote adhesion,morphological changes, and/or extravasation concurrent with chemotaxisthrough the tissues, increase cell proliferation and cytotoxic activity,stimulate antigen presentation and provide other phenotypic changesincluding generation of memory cell types. Immune response if also meantto refer to the activity of immune cells to suppress or regulateinflammatory or cytotoxic activity of other immune cells.

As used herein, the terms “polynucleotide” and “nucleic acid molecule”are used interchangeably to refer to polymeric forms of nucleotides ofany length. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example, single-,double-stranded and triple helical molecules, a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA,recombinant polynucleotides, branched polynucleotides, aptamers,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. A nucleic acid molecule mayalso comprise modified nucleic acid molecules (e.g., comprising modifiedbases, sugars, and/or internucleotide linkers).

The term “polypeptide” is meant to refer to any polymer preferablyconsisting essentially of any of the 20 natural amino acids regardlessof its size. Although the term “protein” is often used in reference torelatively large proteins, and “peptide” is often used in reference tosmall polypeptides, use of these terms in the field often overlaps. Theterm “polypeptide” refers generally to proteins, polypeptides, andpeptides unless otherwise noted. Peptides useful in accordance with thepresent invention in general will be generally between about 0.1 to 100KD or greater up to about 1000 KD, preferably between about 0.1, 0.2,0.5, 1, 2, 5, 10, 20, 30 and 50 KD as judged by standard molecule sizingtechniques such as centrifugation or SDS-polyacrylamide gelelectrophoresis.

The terms “prevent,” “preventing,” “prevention,” “prophylactictreatment” and the like are meant to refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

The term “single chain antibody” is meant to refer to an antibody basedon a single chain format. Single chain antibodies can consist of theminimal binding subunit of antibodies. Single-chain antibodies cancombine only those antigen-binding regions of antibodies on a singlestably-folded polypeptide chain. As such, single-chain antibodies are ofconsiderably smaller size than classical immunoglobulins but retain theantigen-specific binding properties of antibodies. Single chainantibodies may be linked to a wide range of ligands, for exampleeffector molecules or drug conjugates.

The term “soluble” as used herein is meant that the fusion molecule andparticularly a fusion protein that is not readily sedimented under lowG-force centrifugation (e.g. less than about 30,000 revolutions perminute in a standard centrifuge) from an aqueous buffer, e.g., cellmedia. Further, the fusion molecule is soluble if it remains in aqueoussolution at a temperature greater than about 5-37° C. and at or nearneutral pH in the presence of low or no concentration of an anionic ornon-ionic detergent. Under these conditions, a soluble protein willoften have a low sedimentation value e.g., less than about 10 to 50svedberg units.

Aqueous solutions referenced herein typically have a buffering compoundto establish pH, typically within a pH range of about 5-9, and an ionicstrength range between about 2 mM and 500 mM. Sometimes a proteaseinhibitor or mild non-ionic detergent is added. Additionally, a carrierprotein may be added if desired such as bovine serum albumin (BSA) to afew mg/ml. Exemplary aqueous buffers include standard phosphate bufferedsaline, tris-buffered saline, or other well known buffers and cell mediaformulations.

The term “stimulate” or “stimulating” is meant to refer to increase, toamplify, to augment, to boost an immune response. Stimulation can be apositive alteration. An exemplary increase can be e.g., by 5%, 10%, 25%,50%, 75%, or even 90-100%. Other exemplary increases include 2-fold,5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

The term “suppress” or “suppressing” is meant to refer to decrease, toattenuate, to diminish, to arrest, or to stabilize an immune response.Suppression may be a negative alteration. An exemplary decrease can bee.g., by 5%, 10%, 25%, 50%, 75%, or even 90-100%. Exemplary decreasesinclude 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

The term “T-cell Receptor” (TCR) is meant to refer to polypeptides of acomplex of integral membrane proteins that participates in theactivation of T cells in response to the presentation of antigen. Tcells recognize a peptide bound to the MHC product through the αβ.heterodimeric T cell receptor (TCR). The TCR repertoire has extensivediversity created by the same gene rearrangement mechanisms used inantibody heavy and light chain genes [Tonegawa, S. (1988) Biosci. Rep.8:3-26]. Most of the diversity is generated at the junctions of variable(V) and joining (J) (or diversity, D) regions that encode thecomplementarity determining region 3 (CDR3) of the α and β chains [Davisand Bjorkman (1988) Nature 334:395-402]. However, TCRs do not undergosomatic point mutations as do antibodies and, perhaps notcoincidentally. TCRs also do not undergo the same extent of affinitymaturation as antibodies. TCRs as they occur in nature appear to haveaffinities that range from 10⁵ to 10⁷ M.⁻¹ whereas antibodies typicallyhave affinities that range from 10⁵ to 10⁹ M⁻¹ [Davis et al. (1998)Annu. Rev. Immunol. 16:523-544; Eisen et al. (1996) Adv. Protein Chem.49:1-56]. While the absence of somatic mutation in TCRs may beassociated with lower affinities, it has also been argued that there isnot a selective advantage for a TCR to have higher affinity. In fact,the serial-triggering [Valitutti et al. (1995) Nature 375:148-151] andkinetic proofreading [Rabinowitz et al. (1996) Proc. Natl. Acad. Sci.USA 93:1401-1405] models of T cell activation both suggest that longeroff-rates (associated with higher affinity) would be detrimental to thesignaling process. It is also possible that higher affinity TCRs mightnot maintain the peptide specificity required for T cell responses. Forexample, peptides bound within the MHC groove display limited accessiblesurface [Bjorkman, P. J. (1997) Cell 89:167-170], which may in turnlimit the amount of energy that can be generated in the interaction. Onthe other hand, raising the affinity of a TCR by directing the energytoward the MHC helices would presumably lead to thymic deletion duringnegative selection [Bevan, M. J. (1997) Immunity 7:175-178]. The term“TCR” encompasses polyclonal, monoclonal, chimeric, humanized,heterodimeric and single-chain T-cell receptors or functional fragmentthereof, including molecule comprising the TCR Vα and Vβ domains. Theterm “TCR” also encompasses T-cell receptors disclosed in for example,US Provisional Application Entitled “T CELL RECEPTOR FUSIONS ANDCONJUGATES AND METHODS OF USE THEREOF”, filed Mar. 19, 2008 and USPatent Publication US 2003 01-44474A1.

The term “vector” is a nucleic acid molecule that is able to replicateautonomously in a host cell and can accept foreign DNA. A vector carriesits own origin of replication, one or more unique recognition sites forrestriction endonucleases which can be used for the insertion of foreignDNA, and usually selectable markers such as genes coding for antibioticresistance, and often recognition sequences (e.g. promoter) for theexpression of the inserted DNA. Common vectors include plasmid vectorsand phage vectors.

T-Cell Receptors (TCR)

T-cells are a subgroup of cells which together with other immune celltypes (polymorphonuclear, eosinophils, basophils, mast cells, B—, NKcells), constitute the cellular component of the immune system. Underphysiological conditions T-cells function in immune surveillance and inthe elimination of foreign antigen. However, under pathologicalconditions there is compelling evidence that T-cells play a major rolein the causation and propagation of disease. In these disorders,breakdown of T-cell immunological tolerance, either central orperipheral is a fundamental process in the causation of autoimmunedisease.

The TCR is composed of at least seven transmembrane proteins. Thedisulfide-linked (.alpha.beta.) heterodimer forms the monotypic antigenrecognition unit, while the invariant chains of CD3, consisting of.epsilon., .gamma., .delta., and .zeta. and .eta. chains, areresponsible for coupling the ligand binding to signaling pathways thatresult in T-cell activation and the elaboration of the cellular immuneresponses. Despite the gene diversity of the TCR chains, two structuralfeatures are common to all known subunits. Firstly, they aretransmembrane proteins with a single transmembrane spanningdomain—presumably alpha-helical. Secondly, all the TCR chains have theunusual feature of possessing a charged amino acid within the predictedtransmembrane domain. The invariant chains have a single negativecharge, conserved between the mouse and human, and the variant chainspossess one (TCR-beta) or two (TCR-alpha) positive charges. Thetransmembrane sequence of TCR-.alpha. is highly conserved in a number ofspecies and thus phylogenetically may serve an important functionalrole. The octapeptide sequence containing the hydrophilic amino acidsarginine and lysine is identical between the species.

A T-cell response is modulated by antigen binding to a TCR. One type ofTCR is a membrane bound heterodimer consisting of an α and β chainresembling an immunoglobin variable (V) and constant (C) region. The TCRα chain includes a covalently linked V-α and C-α chain, whereas the βchain includes a V-β chain covalently linked to a C-β chain. The V-α andV-β chains form a pocket or cleft that can bind a superantigen orantigen in the context of a major histocompatibility complex (MHC)(known in humans as an HLA complex). See generally Davis Ann. Rev. ofImmunology 3: 537 (1985); Fundamental Immunology 3rd Ed., W. Paul Ed.Rsen Press LTD. New York (1993).

Fusions Proteins

The soluble fusion protein and conjugate molecule complexes of theinvention comprise at least two soluble fusion proteins, where the firstfusion protein comprises a first biologically active polypeptidecovalently linked to interleukin-15 (IL-15) or functional fragmentthereof; and the second fusion protein comprises a second biologicallyactive polypeptide covalently linked to soluble interleukin-15 receptoralpha (IL-15Ra) polypeptide or functional fragment thereof, and whereinIL-15 domain of a first fusion protein binds to the soluble IL-15Radomain of the second fusion protein to form a soluble fusion proteincomplex.

In certain examples, one of the biologically active polypeptidescomprises a first soluble TCR or fragment thereof. The other or secondbiologically active polypeptide comprises the first soluble TCR orfunctional fragment thereof and thus creates a multivalent TCR fusionprotein complex with increased binding activity for cognate ligandscompared to the monovalent TCR. Further, the other biologically activepolypeptide comprises a second soluble TCR or functional fragmentthereof, different than the first soluble TCR. In certain examples, TCRsare produced that have higher affinity, or increased binding affinityfor cognate ligands as compared, for example, to the native TCR. If thesoluble TCR of the invention as described herein has a higher avidity oraffinity for its ligand, then it is useful as a specific probe forcell-surface bound antigen. In certain preferred examples of theinvention, the TCR is specific for recognition of a particular antigen.

In exemplary embodiments, TCR is a heterodimer comprising an α chain(herein referred to as α, alpha or a chain) and a β chain (hereinreferred to as β, beta or b chain). In other exemplary embodiments, theTCR comprises a single chain TCR polypeptide. The single chain TCR maycomprise a TCR V-α chain covalently linked to a TCR V-β chain by apeptide linker sequence. The single chain TCR may further comprise asoluble TCR Cβ chain fragment covalently linked to a TCR V-β chain. Thesingle chain TCR may further comprise a soluble TCR Cα chain fragmentcovalently linked to a TCR V-α chain.

In a further embodiment, one or both of the first and secondbiologically active polypeptides comprises an antibody or functionalfragment thereof.

As used herein, the term “biologically active polypeptide” or “effectormolecule” is meant an amino acid sequence such as a protein, polypeptideor peptide; a sugar or polysaccharide; a lipid or a glycolipid,glycoprotein, or lipoprotein that can produce the desired effects asdiscussed herein. Effector molecules also include chemical agents. Alsocontemplated are effector molecule nucleic acids encoding a biologicallyactive or effector protein, polypeptide, or peptide. Thus, suitablemolecules include regulatory factors, enzymes, antibodies, or drugs aswell as DNA, RNA, and oligonucleotides. The biologically activepolypeptides or effector molecule can be naturally-occurring or it canbe synthesized from known components, e.g., by recombinant or chemicalsynthesis and can include heterologous components. A biologically activepolypeptides or effector molecule is generally between about 0.1 to 100KD or greater up to about 1000 KD, preferably between about 0.1, 0.2,0.5, 1, 2, 5, 10, 20, 30 and 50 KD as judged by standard molecule sizingtechniques such as centrifugation or SDS-polyacrylamide gelelectropheresis. Desired effects of the invention include, but are notlimited to, for example, forming a TCR fusion protein complex withincreased binding activity, killing a target cell, e.g. either to inducecell proliferation or cell death, initiate an immune response, inpreventing or treating a disease, or to act as a detection molecule fordiagnostic purposes. For such detection, an assay could be used, forexample an assay that includes sequential steps of culturing cells toproliferate same, and contacting the cells with a TCR fusion complex ofthe invention and then evaluating whether the TCR fusion complexinhibits further development of the cells.

Covalently linking the effector molecule to the TCR peptide inaccordance with the invention provides a number of significantadvantages. TCR fusion complexes of the invention can be produced thatcontain a single effector molecule, including such a peptide of knownstructure. Additionally, a wide variety of effector molecules can beproduced in similar DNA vectors. That is, a library of differenteffector molecules can be linked to the TCR molecule for presentation ofinfected or diseased cells. Further, for therapeutic applications,rather than administration of an TCR molecule to a subject, a DNAexpression vector coding for the TCR molecule linked to the effectorpeptide can be administered for in vivo expression of the TCR fusioncomplex. Such an approach avoids costly purification steps typicallyassociated with preparation of recombinant proteins and avoids thecomplexities of antigen uptake and processing associated withconventional approaches.

As noted, components of the fusion proteins disclosed herein, e.g.,effector molecule such as cytokines, chemokines, growth factors, proteintoxins, immunoglobulin domains or other bioactive molecules and anypeptide linkers, can be organized in nearly any fashion provided thatthe fusion protein has the function for which it was intended. Inparticular, each component of the fusion protein can be spaced fromanother component by at least one suitable peptide linker sequence ifdesired. Additionally, the fusion proteins may include tags, e.g., tofacilitate modification, identification and/or purification of thefusion protein. More specific fusion proteins are in the Examplesdescribed below.

Linkers

The fusion complexes of the invention preferably also include a flexiblelinker sequence interposed between the IL-15 or IL-15Rα domains and thebiologically active polypeptide. The linker sequence should alloweffective positioning of the biologically active polypeptide withrespect to the IL-15 or IL-15Rα domains to allow functional activity ofboth domains. In embodiments where the biologically active polypeptideis a TCR, the linker sequence positions the TCR molecule binding grooveso that the T cell receptor can recognize presenting MHC-peptidecomplexes and can deliver the effector molecule to a desired site.Successful presentation of the effector molecule can modulate theactivity of a cell either to induce or to inhibit T-cell proliferation,or to initiate or inhibit an immune response to a particular site, asdetermined by the assays disclosed below, including the in vitro assaysthat includes sequential steps of culturing T cells to proliferate same,and contacting the T cells with a TCR fusion complex of the inventionand then evaluating whether the TCR fusion complex inhibits furtherdevelopment of the cells.

In certain embodiments, the soluble fusion protein complex has a linkerwherein the first biologically active polypeptide is covalently linkedto IL-15 (or functional fragment thereof) by polypeptide linkersequence.

In other certain embodiments, the soluble fusion protein complex asdescribed herein has a linker wherein the second biologically activepolypeptide is covalently linked to IL-15Ra polypeptide (or functionalfragment thereof) by polypeptide linker sequence.

The linker sequence is preferably encoded by a nucleotide sequenceresulting in a peptide that can effectively position the binding grooveof the TCR molecule for recognition of a presenting antigen. As usedherein, the phrase “effective positioning of the biologically activepolypeptide with respect to the IL-15 or IL-15Rα domains”, or othersimilar phrase, is intended to mean the biologically active polypeptidelinked to the IL-15 or IL-15Rα domains is positioned so that the IL-15or IL-15Rα domains are capable of interacting with each other to form aprotein complex. In certain embodiments, the IL-15 or IL-15Rα domainsare effectively positioned to allow interactions with immune cells toinitiate or inhibit an immune reaction, or to inhibit orstimulate celldevelopment.

Preferably the linker sequence comprises from about 7 to 20 amino acids,more preferably from about 8 to 16 amino acids. The linker sequence ispreferably flexible so as not hold the biologically active polypeptideor effector molecule in a single undesired conformation. The linkersequence can be used, e.g., to space the recognition site from the fusedmolecule. Specifically, the peptide linker sequence can be positionedbetween the biologically active polypeptide and the effector molecule,e.g., to chemically cross-link same and to provide molecularflexibility. The linker is preferably predominantly comprises aminoacids with small side chains, such as glycine, alanine and serine, toprovide for flexibility. Preferably about 80 or 90 percent or greater ofthe linker sequence comprises glycine, alanine or serine residues,particularly glycine and serine residues. For a fusion protein complexthat comprise a heterodimer TCR, the linker sequence is suitably linkedto the b chain of the TCR molecule, although the linker sequence alsocould be attached to the a chain of the TCR molecule. Alternatively,linker sequence may be linked to both a and b chains of the TCRmolecule. When such a beta peptide chain is expressed along with the achain, the linked TCR-effector peptide should fold resulting in afunctional TCR molecule as generally depicted in FIG. 1. One suitablelinker sequence is ASGGGGSGGG (i.e., Ala Ser Gly Gly Gly Gly Ser Gly GlyGly) (SEQ ID NO: 18), preferably linked to the first amino acid of the bdomain of the TCR. Different linker sequences could be used includingany of a number of flexible linker designs that have been usedsuccessfully to join antibody variable regions together, see Whitlow, M.et al., (1991) Methods: A Companion to Methods in Enzymology 2:97-105.In some examples, for covalently linking an effector molecule to a TCR bchain molecule, the amino sequence of the linker should be capable ofspanning suitable distance from the C-terminal residue of the TCR betachain to the N-terminal residue of the effector molecule.

In general, preparation of the fusion protein complexes of the inventioncan be accomplished by procedures disclosed herein and by recognizedrecombinant DNA techniques involving, e.g., polymerase chainamplification reactions (PCR), preparation of plasmid DNA, cleavage ofDNA with restriction enzymes, preparation of oligonucleotides, ligationof DNA, isolation of mRNA, introduction of the DNA into a suitable cell,transformation or transfection of a host, culturing of the host.Additionally, the fusion molecules can be isolated and purified usingchaotropic agents and well known electrophoretic, centrifugation andchromatographic methods. See generally, Sambrook et al., MolecularCloning: A Laboratory Manual (2nd ed. (1989); and Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York(1989) for disclosure relating to these methods.

As used herein, biologically active polypeptides or effector moleculesof the invention may include factors such as cytokines, chemokines,growth factors, protein toxins, immunoglobulin domains or otherbioactive proteins such as enzymes. Also biologically activepolypeptides may include conjugates to other compounds such asnon-protein toxins, cytotoxic agents, chemotherapeutic agents,detectable labels, radioactive materials and such.

Cytokines of the invention are defined by any factor produced by cellsthat affect other cells and are responsible for any of a number ofmultiple effects of cellular immunity. Examples of cytokines include butare not limited to the IL-2 family, interferon (IFN), IL-10, IL-1,IL-17, TGF and TNF cytokine families, and to IL-1 through IL-35, IFN-α,IFN-β, IFN-γ, TGF-β, TNF-α and TNFβ.

In an aspect of the invention, the first fusion protein comprises afirst biologically active polypeptide covalently linked tointerleukin-15 (IL-15) domain or a functional fragment thereof. IL-15 isa cytokine which affects T-cell activation and proliferation. IL-15activity in affecting immune cell activation and proliferation issimilar in some respects to IL2, although fundamental difference havebeen well characterized (Waldmann, T A, 2006, Nature Rev. Immunol.6:595-601).

In another aspect of the invention, the first fusion protein comprisesan interleukin-15 (IL-15) domain that is an IL-15 variant (also referredto herein as IL-15 mutant). The IL-15 variant preferably comprises adifferent amino acid sequence that the native (or wild type) IL-15protein. The IL-15 variant preferably binds the IL-15Ra polypeptide andfunctions as an IL-15 agonist or antagonist. Preferably IL-15 variantswith agonist activity have super agonist activity. In some embodiments,the IL-15 variant can function as an IL-15 agonist or antagonistindependent of its association with IL-15Ra. IL-15 agonists areexemplified by comparable or increased biological activity compared towild type IL-15. IL-15 antagonists are exemplified by decreasedbiological activity compared to wild type IL-15 or by the ability toinhibit IL-15-mediated responses. In some examples, the IL-15 variantbinds with increased or decreased activity to the IL-15RβγC receptors.In some embodiments, the sequence of the IL-15 variant has at least oneamino acid change, e.g. substitution or deletion, compared to the nativeIL-2 sequence, such changes resulting in IL-15 agonist or antagonistactivity. Preferably the amino acid substitutions/deletions are in thedomains of IL-15 that interact with IL-15Rβ and/or γC. More preferably,the amino acid substitutions/deletions do not affect binding to theIL-15Ra polypeptide or the ability to produce the IL-15 variant.Suitable amino acid substitutions/deletions to generate IL-15 variantscan be identified based on putative or known IL-15 structures,comparisons of IL-15 with homologous molecules such as IL-2 with knownstructure, through rational or random mutagenesis and functional assays,as provided herein, or other empirical methods. Additionally suitableamino acid substitutions can be conservative or non-conservative changesand insertions of additional amino acids Preferably IL-15 variants ofthe invention contain one or more than one amino acidsubstitutions/deletions at position 8, 61, 65, 72, 92, 101, 108, or 111of the mature human IL-15 sequence; particularly, D8N (“D8” refers tothe amino acid and residue position in the native mature human IL-15sequence and “N” refers to the substituted amino acid residue at thatposition in the IL-15 variant), D8A, D61A, N65A, N72R or Q108Asubstitutions result in IL-15 variants with antagonist activity and N72Dsubstitutions result in IL-15 variants with agonist activity.

While in one aspect of the invention the IL-15 variant is a component ofa fusion protein complex, in other aspects the IL-15 variant is anon-fusion protein. Preferably the non-fusion form of the IL-15 variantis a soluble cytokine that functions as an IL-15 agonist or antagonist.In some embodiments, the non-fusion IL-15 variant forms a complex withIL-15Ra whereas in other embodiment it acts independently of IL-15Ra.

Chemokines of the invention, similar to cytokines, are defined as anychemical factor or molecule which when exposed to other cells areresponsible for any of a number of multiple effects of cellularimmunity. Suitable chemokines may include but are not limited to theCXC, CC, C, and CX₃C chemokine families and to CCL-1 through CCL-28,CXC-1 through CXC-17, XCL-1, XCL-2, CX3CL1, MIP-1b, IL-8, MCP-1, andRantes.

Growth factors include any molecules which when exposed to a particularcell induce proliferation and/or differentiation of the affected cell.Growth factors include proteins and chemical molecules, some of whichinclude: GM-CSF, G-CSF, human growth factor and stem cell growth factor.Additional growth factors may also be suitable for uses describedherein.

Toxins or cytotoxic agents include any substance which has a lethaleffect or an inhibitory effect on growth when exposed to cells. Morespecifically, the effector molecule can be a cell toxin of, e.g., plantor bacterial origin such as, e.g., diphtheria toxin (DT), shiga toxin,abrin, cholera toxin, ricin, saporin, pseudomonas exotoxin (PE),pokeweed antiviral protein, or gelonin. Biologically active fragments ofsuch toxins are well known in the art and include, e.g., DT A chain andricin A chain. Additionally, the toxin can be an agent active at thecell surface such as, e.g., phospholipase enzymes (e.g., phospholipaseC).

Further, the effector molecule can be a chemotherapeutic drug such as,e.g., vindesine, vincristine, vinblastin, methotrexate, adriamycin,bleomycin, or cisplatin.

Additionally, the effector molecule can be a detectably-labeled moleculesuitable for diagnostic or imaging studies. Such labels include biotinor streptavidin/avidin, a detectable nanoparticles or crystal, an enzymeor catalytically active fragment thereof, a fluorescent label such asgreen fluorescent protein, FITC, phycoerythrin, cychome, texas red orquantum dots; a radionuclide e.g., iodine-131, yttrium-90, rhenium-188or bismuth-212; a phosphorescent or chemiluminescent molecules or alabel detectable by PET, ultrasound or MRI such as Gd— or paramagneticmetal ion-based contrast agents. See e.g., Moskaug, et al. J. Biol.Chem. 264, 15709 (1989); Pastan, I. et al. Cell 47, 641, 1986; Pastan etal., Recombinant Toxins as Novel Therapeutic Agents, Ann. Rev. Biochem.61, 331, (1992); “Chimeric Toxins” Olsnes and Phil, Pharmac. Ther., 25,355 (1982); published PCT application no. WO 94/29350; published PCTapplication no. WO 94/04689; published PCT application no. WO2005046449and U.S. Pat. No. 5,620,939 for disclosure relating to making and usingproteins comprising effectors or tags.

A protein fusion or conjugate complex that includes a covalently linkedIL-15 and IL-15Ra domains has several important uses. For example, theprotein fusion or conjugate complex comprising a TCR can be employed todeliver the IL-15/IL-15Ra complex\to certain cells capable ofspecifically binding the TCR. Accordingly, the protein fusion orconjugate complex provide means of selectively damaging or killing cellscomprising the ligand. Examples of cells or tissue capable of beingdamaged or killed by the protein fusion or conjugate complexescomprising a TCR include tumors and virally or bacterially infectedcells expressing one or more ligands capable of being specifically boundby the TCR. Cells or tissue susceptible to being damaged or killed canbe readily assayed by the methods disclosed herein.

The IL-15 and IL-15Ra polypeptides of the invention suitably correspondin amino acid sequence to naturally occurring IL-15 and IL-15Ramolecules, e.g. IL-15 and IL-15Ra molecules of a human, mouse or otherrodent, or other mammal.

In some settings it can be useful to make the protein fusion orconjugate complexes of the present invention polyvalent, e.g., toincrease the valency of the sc-TCR. In particular, interactions betweenthe IL-15 and IL-15Ra domains of the fusion protein complex provide ameans of generating polyvalent complexes. In addition, the polyvalentfusion protein can made by covalently or non-covalently linking togetherbetween one and four proteins (the same or different) by using e.g.,standard biotin-streptavidin labeling techniques, or by conjugation tosuitable solid supports such as latex beads. Chemically cross-linkedproteins (for example cross-linked to dendrimers) are also suitablepolyvalent species. For example, the protein can be modified byincluding sequences encoding tag sequences that can be modified such asthe biotinylation BirA tag or amino acid residues with chemicallyreactive side chains such as Cys or His. Such amino acid tags orchemically reactive amino acids may be positioned in a variety ofpositions in the fusion protein, preferably distal to the active site ofthe biologically active polypeptide or effector molecule. For example,the C-terminus of a soluble fusion protein can be covalently linked to atag or other fused protein which includes such a reactive amino acid(s).Suitable side chains can be included to chemically link two or morefusion proteins to a suitable dendrimer or other nanoparticle to give amultivalent molecule. Dendrimers are synthetic chemical polymers thatcan have any one of a number of different functional groups of theirsurface (D. Tomalia, Aldrichimica Acta, 26:91:101 (1993)). Exemplarydendrimers for use in accordance with the present invention include e.g.E9 starburst polyamine dendrimer and E9 combust polyamine dendrimer,which can link cystine residues.

Nucleic Acids and Vectors

Nucleic Acids

The invention further provides nucleic acid sequences and particularlyDNA sequences that encode the present fusion proteins. Preferably, theDNA sequence is carried by a vector suited for extrachromosomalreplication such as a phage, virus, plasmid, phagemid, cosmid, YAC, orepisome. In particular, a DNA vector that encodes a desired fusionprotein can be used to facilitate preparative methods described hereinand to obtain significant quantities of the fusion protein. The DNAsequence can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. A variety ofhost-vector systems may be utilized to express the protein-codingsequence. These include mammalian cell systems infected with virus(e.g., vaccinia virus, adenovirus, etc.); insect cell systems infectedwith virus (e.g., baculovirus); microorganisms such as yeast containingyeast vectors, or bacteria transformed with bacteriophage DNA, plasmidDNA or cosmid DNA. Depending on the host-vector system utilized, any oneof a number of suitable transcription and translation elements may beused. See generally Sambrook et al., supra and Ausubel et al. supra.

Included in the invention are methods for making a soluble fusionprotein complex, the method comprising introducing into a host cell aDNA vector as described herein encoding the first and second fusionproteins, culturing the host cell in media under conditions sufficientto express the fusion proteins in the cell or the media and allowassociation between IL-15 domain of a first fusion protein and thesoluble IL-15Ra domain of a second fusion protein to form the solublefusion protein complex, purifying the soluble fusion protein complexfrom the host cells or media.

In general, a preferred DNA vector according to the invention comprisesa nucleotide sequence linked by phosphodiester bonds comprising, in a 5′to 3′ direction a first cloning site for introduction of a firstnucleotide sequence encoding a TCR chain, operatively linked to asequence encoding an effector molecule.

The fusion protein components encoded by the DNA vector can be providedin a cassette format. By the term “cassette” is meant that eachcomponent can be readily substituted for another component by standardrecombinant methods. In particular, a DNA vector configured in acassette format is particularly desirable when the encoded fusioncomplex is to be used against pathogens that may have or have capacityto develop serotypes.

To make the vector coding for a TCR fusion complex, the sequence codingfor the TCR molecule is linked to a sequence coding for the effectorpeptide by use of suitable ligases. DNA coding for the presentingpeptide can be obtained by isolating DNA from natural sources such asfrom a suitable cell line or by known synthetic methods, e.g. thephosphate triester method. See, e.g., Oligonucleotide Synthesis, IRLPress (M. J. Gait, ed., 1984). Synthetic oligonucleotides also may beprepared using commercially available automated oligonucleotidesynthesizers. Once isolated, the gene coding for the TCR molecule can beamplified by the polymerase chain reaction (PCR) or other means known inthe art. Suitable PCR primers to amplify the TCR peptide gene may addrestriction sites to the PCR product. The PCR product preferablyincludes splice sites for the effector peptide and leader sequencesnecessary for proper expression and secretion of the TCR-effector fusioncomplex. The PCR product also preferably includes a sequence coding forthe linker sequence, or a restriction enzyme site for ligation of such asequence.

The fusion proteins described herein are preferably produced by standardrecombinant DNA techniques. For example, once a DNA molecule encodingthe TCR protein is isolated, sequence can be ligated to another DNAmolecule encoding the effector polypeptide. The nucleotide sequencecoding for a TCR molecule may be directly joined to a DNA sequencecoding for the effector peptide or, more typically, a DNA sequencecoding for the linker sequence as discussed herein may be interposedbetween the sequence coding for the TCR molecule and the sequence codingfor the effector peptide and joined using suitable ligases. Theresultant hybrid DNA molecule can be expressed in a suitable host cellto produce the TCR fusion complex. The DNA molecules are ligated to eachother in a 5′ to 3′ orientation such that, after ligation, thetranslational frame of the encoded polypeptides is not altered (i.e.,the DNA molecules are ligated to each other in-frame). The resulting DNAmolecules encode an in-frame fusion protein.

Other nucleotide sequences also can be included in the gene construct.For example, a promoter sequence, which controls expression of thesequence coding for the TCR peptide fused to the effector peptide, or aleader sequence, which directs the TCR fusion complex to the cellsurface or the culture medium, can be included in the construct orpresent in the expression vector into which the construct is inserted.An immunoglobulin or CMV promoter is particularly preferred.

In obtaining variant TCR coding sequences, those of ordinary skill inthe art will recognize that TCR-derived proteins may be modified bycertain amino acid substitutions, additions, deletions, andpost-translational modifications, without loss or reduction ofbiological activity. In particular, it is well-known that conservativeamino acid substitutions, that is, substitution of one amino acid foranother amino acid of similar size, charge, polarity and conformation,are unlikely to significantly alter protein function. The 20 standardamino acids that are the constituents of proteins can be broadlycategorized into four groups of conservative amino acids as follows: thenonpolar (hydrophobic) group includes alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine; the polar(uncharged, neutral) group includes asparagine, cysteine, glutamine,glycine, serine, threonine and tyrosine; the positively charged (basic)group contains arginine, histidine and lysine; and the negativelycharged (acidic) group contains aspartic acid and glutamic acid.Substitution in a protein of one amino acid for another within the samegroup is unlikely to have an adverse effect on the biological activityof the protein.

Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50.degree. C., 6.times.SSC (sodium chloride/sodiumcitrate buffer) and 0.1% SDS (sodium dodecyl sulfate) indicate about60-70% homology, hybridization and wash conditions of 50-65.degree. C.,1.times.SSC and 0.1% SDS indicate about 82-97% homology, andhybridization and wash conditions of 52.degree. C., 0.1.times.SSC and0.1% SDS indicate about 99-100% homology. A wide range of computerprograms for comparing nucleotide and amino acid sequences (andmeasuring the degree of homology) are also available, and a listproviding sources of both commercially available and free software isfound in Ausubel et al. (1999). Readily available sequence comparisonand multiple sequence alignment algorithms are, respectively, the BasicLocal Alignment Search Tool (BLAST) (Altschul et al., 1997) and ClustalWprograms. BLAST is available on the world wide web at ncbi.nlm.nih.govand a version of ClustalW is available at 2.ebi.ac.uk.

The components of the fusion protein can be organized in nearly anyorder provided each is capable of performing its intended function. Forexample, in one embodiment, the TCR is situated at the C or N terminalend of the effector molecule.

Preferred effector molecules of the invention will have sizes conduciveto the function for which those domains are intended. The effectormolecules of the invention can be made and fused to the TCR by a varietyof methods including well-known chemical cross-linking methods. Seee.g., Means, G. E. and Feeney, R. E. (1974) in Chemical Modification ofProteins, Holden-Day. See also, S. S. Wong (1991) in Chemistry ofProtein Conjugation and Cross-Linking, CRC Press. However it isgenerally preferred to use recombinant manipulations to make thein-frame fusion protein.

As noted, a fusion molecule or a conjugate molecule in accord with theinvention can be organized in several ways. In an exemplaryconfiguration, the C-terminus of the TCR is operatively linked to theN-terminus of the effector molecule. That linkage can be achieved byrecombinant methods if desired. However, in another configuration, theN-terminus of the TCR is linked to the C-terminus of the effectormolecule.

Alternatively, or in addition, one or more additional effector moleculescan be inserted into the TCR fusion or conjugate complexes as needed.

Vectors and Expression

A number of strategies can be employed to express protein fusioncomplexes of the invention. For example, the TCR gene fusion constructdescribed above can be incorporated into a suitable vector by knownmeans such as by use of restriction enzymes to make cuts in the vectorfor insertion of the construct followed by ligation. The vectorcontaining the gene construct is then introduced into a suitable hostfor expression of the TCR fusion peptide. See, generally, Sambrook etal., supra. Selection of suitable vectors can be made empirically basedon factors relating to the cloning protocol. For example, the vectorshould be compatible with, and have the proper replicon for the hostthat is being employed. Further the vector must be able to accommodatethe DNA sequence coding for the TCR fusion complex that is to beexpressed. Suitable host cells include eukaryotic and prokaryotic cells,preferably those cells that can be easily transformed and exhibit rapidgrowth in culture medium. Specifically preferred hosts cells includeprokaryotes such as E. coli, Bacillus subtillus, etc. and eukaryotessuch as animal cells and yeast strains, e.g., S. cerevisiae. Mammaliancells are generally preferred, particularly J558, NSO, SP2-O or CHO.Other suitable hosts include, e.g., insect cells such as Sf9.Conventional culturing conditions are employed. See Sambrook, supra.Stable transformed or transfected cell lines can then be selected. Cellsexpressing a TCR fusion complex of the invention can be determined byknown procedures. For example, expression of a TCR fusion complex linkedto an immunoglobulin can be determined by an ELISA specific for thelinked immunoglobulin and/or by immunoblotting. Other methods fordetecting expression of fusion proteins comprising TCRs linked to IL-15or IL-15Ra domains are disclosed in the Examples.

As mentioned generally above, a host cell can be used for preparativepurposes to propagate nucleic acid encoding a desired fusion protein.Thus a host cell can include a prokaryotic or eukaryotic cell in whichproduction of the fusion protein is specifically intended. Thus hostcells specifically include yeast, fly, worm, plant, frog, mammaliancells and organs that are capable of propagating nucleic acid encodingthe fusion. Non-limiting examples of mammalian cell lines which can beused include CHO dhfr-cells (Urlaub and Chasm, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)), 293 cells (Graham et al., J Gen. Virol., 36:59(1977)) or myeloma cells like SP2 or NSO (Galfre and Milstein, Meth.Enzymol., 73(B):3 (1981)).

Host cells capable of propagating nucleic acid encoding a desired fusionprotein encompass non-mammalian eukaryotic cells as well, includinginsect (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P.pastoris., K. lactis, H. polymorpha; as generally reviewed by Fleer, R.,Current Opinion in Biotechnology, 3(5):486496 (1992)), fungal and plantcells. Also contemplated are certain prokaryotes such as E. coli andBacillus.

Nucleic acid encoding a desired fusion protein can be introduced into ahost cell by standard techniques for transfecting cells. The term“transfecting” or “transfection” is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection, viraltransduction and/or integration. Suitable methods for transfecting hostcells can be found in Sambrook et al. supra, and other laboratorytextbooks.

Various promoters (transcriptional initiation regulatory region) may beused according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

Promoter selection is also dependent upon the desired efficiency andlevel of peptide or protein production. Inducible promoters such as tacare often employed in order to dramatically increase the level ofprotein expression in E. coli. Overexpression of proteins may be harmfulto the host cells. Consequently, host cell growth may be limited. Theuse of inducible promoter systems allows the host cells to be cultivatedto acceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

Various signal sequences may be used according to the invention. Asignal sequence which is homologous to the TCR coding sequence may beused. Alternatively, a signal sequence which has been selected ordesigned for efficient secretion and processing in the expression hostmay also be used. For example, suitable signal sequence/host cell pairsinclude the B. subtilis sacB signal sequence for secretion in B.subtilis, and the Saccharomyces cerevisiae .alpha.-mating factor or P.pastoris acid phosphatase phoI signal sequences for P. pastorissecretion. The signal sequence may be joined directly through thesequence encoding the signal peptidase cleavage site to the proteincoding sequence, or through a short nucleotide bridge consisting ofusually fewer than ten codons, where the bridge ensures correct readingframe of the downstream TCR sequence.

Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptII confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His.sup.-S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 .mu.g of the antibiotic/mL of medium.

The expression construct is assembled by employing known recombinant DNAtechniques (Sambrook et al., 1989; Ausubel et al., 1999). Restrictionenzyme digestion and ligation are the basic steps employed to join twofragments of DNA. The ends of the DNA fragment may require modificationprior to ligation, and this may be accomplished by filling in overhangs,deleting terminal portions of the fragment(s) with nucleases (e.g.,ExoIII), site directed mutagenesis, or by adding new base pairs by PCR.Polylinkers and adaptors may be employed to facilitate joining ofselected fragments. The expression construct is typically assembled instages employing rounds of restriction, ligation, and transformation ofE. coli. Numerous cloning vectors suitable for construction of theexpression construct are known in the art (.lambda.ZAP and pBLUESCRIPTSK-1, Stratagene, LaJolla, Calif., pET, Novagen Inc., Madison,Wis.—cited in Ausubel et al., 1999) and the particular choice is notcritical to the invention. The selection of cloning vector will beinfluenced by the gene transfer system selected for introduction of theexpression construct into the host cell. At the end of each stage, theresulting construct may be analyzed by restriction, DNA sequence,hybridization and PCR analyses.

The expression construct may be transformed into the host as the cloningvector construct, either linear or circular, or may be removed from thecloning vector and used as is or introduced onto a delivery vector. Thedelivery vector facilitates the introduction and maintenance of theexpression construct in the selected host cell type. The expressionconstruct is introduced into the host cells by any of a number of knowngene transfer systems (e.g., natural competence, chemically mediatedtransformation, protoplast transformation, electroporation, biolistictransformation, transfection, or conjugation) (Ausubel et al., 1999;Sambrook et al., 1989). The gene transfer system selected depends uponthe host cells and vector systems used.

For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

The present invention further provides a production process forisolating a fusion protein of interest. In the process, a host cell(e.g., a yeast, fungus, insect, bacterial or animal cell), into whichhas been introduced a nucleic acid encoding the protein of the interestoperatively linked to a regulatory sequence, is grown at productionscale in a culture medium in the presence of the fusion protein tostimulate transcription of the nucleotides sequence encoding the fusionprotein of interest. Subsequently, the fusion protein of interest isisolated from harvested host cells or from the culture medium. Standardprotein purification techniques can be used to isolate the protein ofinterest from the medium or from the harvested cells. In particular, thepurification techniques can be used to express and purify a desiredfusion protein on a large-scale (i.e. in at least milligram quantities)from a variety of implementations including roller bottles, spinnerflasks, tissue culture plates, bioreactor, or a fermentor.

An expressed protein fusion complex can be isolated and purified byknown methods. Typically the culture medium is centrifuged and then thesupernatant is purified by affinity or immunoaffinity chromatography,e.g. Protein-A or Protein-G affinity chromatography or an immunoaffinityprotocol comprising use of monoclonal antibodies that bind the expressedfusion complex such as a linked TCR or immunoglobulin region thereof.The fusion proteins of the present invention can be separated andpurified by appropriate combination of known techniques. These methodsinclude, for example, methods utilizing solubility such as saltprecipitation and solvent precipitation, methods utilizing thedifference in molecular weight such as dialysis, ultra-filtration,gel-filtration, and SDS-polyacrylamide gel electrophoresis, methodsutilizing a difference in electrical charge such as ion-exchange columnchromatography, methods utilizing specific affinity such as affinitychromatograph, methods utilizing a difference in hydrophobicity such asreverse-phase high performance liquid chromatograph and methodsutilizing a difference in isoelectric point, such as isoelectricfocusing electrophoresis, metal affinity columns such as Ni-NTA. Seegenerally Sambrook et al. and Ausubel et al. supra for disclosurerelating to these methods.

It is preferred that the fusion proteins of the present invention besubstantially pure. That is, the fusion proteins have been isolated fromcell substituents that naturally accompany it so that the fusionproteins are present preferably in at least 80% or 90% to 95%homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity(w/w) are most preferred for many pharmaceutical, clinical and researchapplications. Once substantially purified the fusion protein should besubstantially free of contaminants for therapeutic applications. Oncepurified partially or to substantial purity, the soluble fusion proteinscan be used therapeutically, or in performing in vitro or in vivo assaysas disclosed herein. Substantial purity can be determined by a varietyof standard techniques such as chromatography and gel electrophoresis.

Truncated TCR fusion complexes of the invention contain a TCR moleculethat is sufficiently truncated so the TCR fusion complex can be secretedinto culture medium after expression. Thus, a truncated TCR fusioncomplex will not include regions rich in hydrophobic residues, typicallythe transmembrane and cytoplasmic domains of the TCR molecule. Thus, forexample, for a preferred truncated DR1 TCR molecule of the invention,preferably from about residues 199 to 237 of the b chain and from aboutresidues 193 to 230 of the a chain of the TCR molecule are not includedin the truncated TCR fusion complex.

The present TCR fusion and conjugate complexes are suitable for in vitroor in vivo use with a variety of cells that are infected or that maybecome infected by one or more diseases.

Methods

Therapeutic

Included in the invention are methods for preventing or treating diseasein a patient in which the diseased cells express a disease associatedantigen, the method comprising administering to the patient a solublefusion protein complex comprising a biologically active polypeptiderecognizing a disease-associated antigen, forming a specific bindingcomplex (bridge) between antigen-expressing diseased cells andIL-15R-expressing immune cells sufficient to localize the immune cells,and damaging or killing the disease cells sufficient to prevent or treatthe disease in the patient.

Included are methods for preventing or treating disease in a patient inwhich the diseased cells express a disease associated antigen, themethod comprising mixing immune cells bearing the IL-15R chains with asoluble fusion protein complex comprising a biologically activepolypeptide recognizing a disease-associated antigen, for example apeptide/MHC complex, administering to the patient the immune cell-fusionprotein complex mixture, forming a specific binding complex (bridge)between antigen-expressing diseased cells and IL-15R-expressing immunecells sufficient to localize the immune cells, and damaging or killingthe disease cells sufficient to prevent or treat the disease in thepatient.

Also included in the invention are methods for killing a target cell,the method comprising contacting a plurality of cells with a solublefusion protein complex, where the plurality of cells further comprisesimmune cells bearing the IL-15R chains recognized by the IL-15 domain ofclaim 1 and the target cells bearing an antigen recognized by at leastone of the biologically active polypeptides as described herein, forminga specific binding complex (bridge) between the antigen on the targetcells and the IL-15R chains on the immune cells sufficient to bind andactivate the immune cells; and killing the target cells by the boundactivated immune cells.

Also included in the inventions are methods to increase in vivo halflife of IL-15 and/or enhance its ability to stability bind immune cells(e.g. increase cell surface residency time) through generation of asoluble fusion protein complex. For example, evaluation of thepharmacokinetic parameters and cell surface residency time of the fusionprotein complex are conducted and compared to IL-15, as describedherein. Fusion protein complexes with an increased serum half life orcell surface residency time are preferable as based on their improvedtherapeutic utility.

Examples of diseases that can be treated include, but are not limitedto, neoplasia, including cancer, or viral infection. By “neoplasia” ismeant any disease that is caused by or results in inappropriately highlevels of cell division, inappropriately low levels of apoptosis, orboth. For example, cancer is an example of a neoplasia. Examples ofcancers include, without limitation, leukemias (e.g., acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

Also included are methods of stimulating immune responses in a mammalcomprising administering to the mammal an effective amount of thesoluble fusion protein complex or IL-15 variant as described herein.Also included are methods of suppressing immune responses in a mammalcomprising administering to the mammal an effective amount of thesoluble fusion protein complex or IL-15 variant as described herein. Inthe case of immune suppression, a fusion protein complex or IL-15variant comprising IL-15 antagonists or IL-15 domains that lack theability to bind the IL-15βγ_(c) complex may be particularlyadvantageous.

As an illustration of the use of the fusion protein complextherapeutics, a cultured cell can be infected by a pathogen of a singleserotype. The infected cell is then contacted by a specified fusionprotein complex in vitro. As discussed previously, the fusion proteincomplex is configured so that the toxic domain is presented to theinfected cell by the association of the TCR. After providing forintroduction of the bioactive molecule to the cell (generally less thanabout 30 minutes), the cells are allowed to cause a desired effect for atime period of about up to about 2 to 24 hours, typically about 18hours. After this time, the cells are washed in a suitable buffer orcell medium and then evaluated for viability. The time allotted for cellkilling or injury by the fusion protein complex will vary with theparticular effector molecule chosen. However viability can often beassessed after about 2 to 6 hours up to about 24 hours. As will beexplained in more detail below, cell viability can be readily measuredand quantified by monitoring uptake of certain well-known dyes (e.g.,trypan blue) or fluors.

Cells incubated with the fusion protein complex of the present inventioncan be assayed for viability by standard methods. In one exemplaryapproach, cell viability can be readily assayed by measuring DNAreplication following or during incubation. For example, a preferredassay involves cell uptake of one or more detectably labeled nucleosidessuch as radiolabelled thymidine. The uptake can be conveniently measuredby several conventional approaches including trichloroacetic acid (TCA)precipitation followed by scintillation counting. Other cell viabilitymethods include well-known trypan blue exclusion techniques orWST-1-based proliferation assays.

The TCR molecules of the fusion complexes of the invention suitablycorrespond in amino acid sequence to naturally occurring TCR molecules,e.g. TCR molecules of a human, mouse or other rodent, or other mammal.

Accordingly, one treatment method of the invention for inhibition of anautoimmune or inflammatory response would include a fusion proteincomplex which comprises a T cell receptor or antibody with bindingspecificity to a disease associated antigen. Preferably, a “truncated”soluble TCR complex is administered, i.e. the TCR complex does notcontain a transmembrane portion. The fusion protein complex alsocomprises an IL-15 variants that functions as an IL-15 antagonist tosuppress the unwanted immune response. Follow administration, the TCR orantibody domain targets the fusion protein complex to the disease sitewhere the IL-15 antagonist suppresses the autoimmune or inflammatoryresponse. Such fusion protein complex may particularly useful fortreatment of allergies, autoimmune diseases such as multiple sclerosis,insulin-dependent diabetes mellitus and rheumatoid arthritis ortransplant rejection. Similar non-targeted approaches could be carriedout using antagonist IL-15 variants as non-fusion proteins.

Another treatment method of the invention for induction of an immuneresponse provides for the administration of an effective amount of aprotein fusion complexes of the invention in the presence of anycostimulatory effector molecule such as a cytokine to thereby induce adesired immune response at the location of the presented antigen whichbinds the biologically active polypeptide.

Different therapies of the invention also may be used in combination aswell as with other known therapeutic agents such as anti-inflammatorydrugs to provide a more effective treatment of a disorder. For example,immunosuppressive protein fusion complexes or IL-15 variants can be usedin combination with anti-inflammatory agents such as corticosteroids andnonsteroidal drugs for the treatment of autoimmune disorders andallergies.

Compounds of the invention will be especially useful to a human patientwho has or is suspected of having a malignant disease, disorder orcondition. Compounds of the invention will be particularly useful intargeting particular tumor antigens in human patients. Specific examplesof diseases which may be treated in accordance with the inventioninclude cancers, e.g. breast, prostate, etc, viral infections, e.g. HCV,HIV, etc. as well as other specific disorders of conditions mentionedherein.

Without wishing to be bound by theory, it is believed the multiple anddistinct covalently linked compounds of this invention (i.e. at leastIL-15 in combination with at least one TCR) can significantly enhanceefficacy of the IL-15, e.g., by increasing targeting of IL-15 to targetantigen in subject individuals.

Moreover, by virtue of the covalent linkage, the conjugates of theinvention present the IL-15 and the TCR to the subject cell essentiallysimultaneously, an effect that may not be readily achieved byadministering the same compounds in a drug “cocktail” formulationwithout covalently linking the compounds.

It also has been reported that treatment with treatment with one drugcan in turn sensitize a patient to another drug. Accordingly, theessentially simultaneous presentation to the subject cell of IL-15 andTCR via a conjugate of the invention may enhance drug activity, e.g., byproviding synergistic results and/or by enhancing production an immuneresponse.

Diagnostic

High affinity or multivalent TCR proteins specific for a particular pMHCligand are useful in diagnosing animals, including humans believed to besuffering from a disease associated with the particular pMHC. The fusionprotein complexes of the present invention are useful for detectingessentially any antigen, including but not limited to, those associatedwith a neoplastic condition, an abnormal protein, an autoimmune diseaseor an infection or infestation with a bacterium, a fungus, a virus, aprotozoan, a yeast, a nematode or other parasite.

In one such method for detecting a tumor or virally infected cell ortissue in a subject, wherein the cell or tissue comprises a tumor orvirus-associated peptide antigen presented on the cells or tissue in thecontext of an MHC complex, comprises: a) administering to the subject asoluble fusion protein complex of the invention comprising a soluble TCRunder conditions that form a specific binding complex between thepresented peptide antigen and the TCR; and b) detecting the specificbinding complex as being indicative of a tumor or virally infected cellor tissue comprising the presented tumor or viral-associated peptideantigen.

The fusion protein complexes can also be used in the diagnosis ofcertain genetic disorders in which there is an abnormal proteinproduced. Exemplary applications for fusion protein complexes are in thediagnosis and treatment of autoimmune diseases in which there is a knownpMHC. Type I diabetes is relatively well characterized with respect tothe autoantigens which attract immune destruction. Multiple sclerosis,celiac disease, inflammatory bowel disease, Crohn's disease andrheumatoid arthritis are additional candidate diseases for suchapplication.

The fusion protein complexes of the present invention comprising IL-15variant polypeptides may be particularly useful in these applications.For example, for a fusion protein complex comprising TCR molecules,interactions between the IL-15 variant domain and the IL-15Rapolypeptide generate multivalent TCR molecules with enhanced antigenbinding activity, as disclosed herein. Moreover, the IL-15 variantcontains amino acid changes that potentially eliminate binding to cellsbearing IL-15RβγC receptors, thereby reducing non-specific ornon-targeted binding to immune cells. As a results, improved detectionof TCR-specific antigens can be achieved with such fusion proteincomplexes. Additionally fusion protein complexes of the invention can befurther multimerized via peptide tags sequences or conjugation todetectable labels, as disclosed herein.

Dosage and Administration

Administration of compounds of the invention may be made by a variety ofsuitable routes including oral, topical (including transdermal, buccalor sublingal), nasal and parenteral (including intraperitoneal,subcutaneous, intravenous, intradermal or intramuscular injection) withoral or parenteral being generally preferred. It also will beappreciated that the preferred method of administration and dosageamount may vary with, for example, the condition and age of therecipient.

Compounds of the invention may be used in therapy alone or inconjunction with other medicaments such those with recognizedpharmacological activity to treat the desired indications. Exemplarymedicaments include recognized therapeutics such as surgery, radiation,chemotherapy and other forms of immunotherapy (e.g. vaccines, antibodybased therapies). The compounds of this invention can be administeredbefore, during or after such therapies as needed.

While one or more compounds of the invention may be administered alone,they also may be present as part of a pharmaceutical composition inmixture with conventional excipient, i.e., pharmaceutically acceptableorganic or inorganic carrier substances suitable for parenteral, oral orother desired administration and which do not deleteriously react withthe active compounds and are not deleterious to the recipient thereof.Pharmaceutical compositions of the invention in general comprise one ormore fusion protein complex or IL-15 variant of the invention or DNAconstructs coding for such compounds together with one or moreacceptable carriers. The carriers must be “acceptable” in the sense ofbeing compatible with other ingredients of the formulation and notdeleterious to the recipient thereof. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

For parenteral application, particularly suitable are solutions,preferably oily or aqueous solutions as well as suspensions, emulsions,or implants, including suppositories. Ampules are convenient unitdosages.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

Therapeutic compounds of the invention also may be incorporated intoliposomes. The incorporation can be carried out according to knownliposome preparation procedures, e.g. sonication and extrusion. Suitableconventional methods of liposome preparation are also disclosed in e.g.A. D. Bangham et al., J. Mol. Biol., 23:238-252 (1965); F. Olson et al.,Biochim. Biophys. Acta, 557:9-23 (1979); F. Szoka et al., Proc. Nat.Acad. Sci., 75:4194-4198 (1978); S. Kim et al., Biochim. Biophys. Acta,728:339-348 (1983); and Mayer et al., Biochim. Biophys. Acta,858:161-168 (1986).

The invention also provides methods for invoking an immune response in amammal such as a human, including vaccinating a mammal such as a humanagainst an infectious agent or a targeted disorder such as cancer.

These methods comprise administering to a mammal an effective amount ofa DNA sequence that comprises a DNA vector that codes for a fusionprotein complex or IL-15 variant of the invention. Preparation ofexpression vectors of fusion protein complexes and IL-15 variants isdescribed above and in the Examples which follow. Methods foradministration of plasmid DNA, uptake of that DNA by cells of theadministered subject and expression of protein has been reported. SeeUlmer, J. B., et al., Science (1993) 259: 1745-1749.

DNA vectors that encode fusion protein complexes and IL-15 variants ofthe invention are suitably administered to a mammal including a humanpreferably by intramuscle injection. Administration of cDNA to skeletalmuscle of a mammal with subsequent uptake of administered expressionvector by the muscle cells and expression of protein encoded by the DNAhas been described by Ulmer et al. and represents an exemplary protocol[Ulmer, J. B., et al., Science 259: 1745-1749]. The optimal dose for agiven therapeutic application can be determined by conventional means.

In addition to treatment of human disorders, fusion protein complexesand IL-15 variants of the invention and DNA constructs of the inventionthat encode such molecules will have significant use for veterinaryapplications, e.g., treatment of disorders of livestock such as cattle,sheep, etc. and pets such as dog and cats.

It will be appreciated that actual preferred amounts of a given fusionprotein complex and IL-15 variant of the invention or DNA constructcoding for same used in a given therapy will vary according to theparticular active compound or compounds being utilized, the particularcompositions formulated, the mode of application, the particular site ofadministration, the patient's weight, general health, sex, etc., theparticular indication being treated, etc. and other such factors thatare recognized by those skilled in the art including the attendantphysician or veterinarian. Optimal administration rates for a givenprotocol of administration can be readily determined by those skilled inthe art using conventional dosage determination tests conducted e.g.with regard to the foregoing guidelines and the assays disclosed herein.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Example 1 Design of a Fusion Protein Complex Comprising scTCR/huIL15 andscTCR/huIL15Rα Fusion Proteins

It has been established that the IL-15 stably binds to the extracellulardomain of the IL-15Rα and that the resulting complex is capable ofmodulating (i.e. either stimulating or blocking) immune responses viathe intermediate or high affinity IL-15R complex (Mortier, E. et al.,2006, J. Biol. Chem., 281: 1612-1619; Stoklasek, T. et al., 2006, JImmunol 177: 6072-6080; Rubinstein, M. P. et al., 2006, Proc Natl AcadSci U S A 103: 9166-9171, Waldmann, T. A., 2006, Nat Rev Immunol 6:595-601). In addition, it has been demonstrated that single-chain TCR orantibody polypeptides can be fused to cytokines and other immuneeffector domains and that such bispecific fusion molecules retainfunctional activity of both fusion domains (Belmont, H. J. et al., 2006,Clin Immunol 121: 29-39; Card, K. F. et al., 2004, Cancer ImmunolImmunother 53: 345-357; Mosquera, L. A. et al., 2005, J Immunol 174:4381-4388; Penichet, M. L., 1997, Hum Antibodies 8: 106-118). Further,it has been shown that multivalent forms of the TCR provide enhancedbinding to their ligands (Zhu, X., H. J., 2006, J Immunol 176:3223-3232). Therefore a feature of the invention provides for a fusionprotein complex comprising at least one fusion protein wherein a firstTCR polypeptide is fused to IL-15 and at least one fusion wherein asecond TCR polypeptide is fused to the extracellular domain of IL-15Rα,such that the two fusion proteins form a complex through bindinginteractions between the IL-15 and IL-15Rα domains. In such a fusionprotein complex, the TCR polypeptides can be the same or different andin either single-chain or heterodimeric format.

An example of a fusion protein complex containing single-chain TCRpolypeptides is shown schematically in FIG. 1A. In this fusion proteincomplex, the multivalent TCR domains provide increased bindingavidity/affinity for their ligands. Exemplary ligands include, but arenot limited to, peptide/MHC complexes. The IL-15/IL-15Rα domains provideimmunomodulatory activity. Representative fusion protein constructscomprising the fusion protein complex are schematically shown in FIG.1B. In these constructs the TCR polypeptide is a single-chain TCR(264scTCR) comprised of TCR-Vα and TCR-Vβ-Cβ domains linked by a peptidelinker sequence ((G₄S)₄) (SEQ ID NO: 3). The scTCR polypeptide is fusedto either the IL-15 or IL-15Rα domains, directly or via a peptide linkersequence. Proceeding the scTCR polypeptide is a signal peptide (orleader peptide) sequence that permits soluble expression. The signalpeptide is subsequently cleaved during protein transport to generate themature fusion protein. In other examples of the fusion protein complex,an antibody domain can substitute a TCR domain depicted in FIGS. 1A and1B. Such an antibody can be in a single-chain or heteromultimericformat. For any of the fusion protein complexes described above,sequences can be human or non-human, for example, but not limited tomouse. These sequences can be employed for part or all of the fusionprotein domains. In addition, the arrangement of the domains can vary solong as the fusion proteins remain soluble and functional.

Example 2 Construction of the c264scTCR/huIL15 Gene Fusion in anExpression Vector

Isolation and characterization of TCR genes for the p53(aa264-272)-specific TCR were described previously (Belmont, H. J. etal., 2006, Clin Immunol 121: 29-39; Card, K. F. et al., 2004, CancerImmunol Immunother 53: 345-357; Mosquera, L. A. et al., 2005, J Immunol174: 4381-4388). To obtain the human IL15 and IL15Rα genes, human PBMCwere isolated from 200 mL of blood of a donor (Lot#2238789, CommunityBlood Bank, Miami, Fla.) with HISTOPAGUE-1077 (Sigma). The cells(1.5×10⁷) were activated by 30 ng/ml of PMA (Sigma), 200 ng/ml ofionomycin, and 220 ng/ml of recombinant human IL2 in IMDM containing10%FBS in a CO₂ incubator for 10 days. The activated cells (1×10⁷ permL) were frozen at −70 C for the further applications. To purify thetotal RNA from the activated PBMC, RNEASY PLUS MINI (Qiagen) was usedaccording to the manufacturer's protocol. Human IL15 gene containing thecoding region and a portion of 5′ and 3′ flanking regions was amplifiedfrom the total RNA with the front primer

(SEQ ID NO: 19) 5′-CACCTTGCCATAGCCAGCTCTTC-3′and the back primer

(SEQ ID NO: 20) 5′-GTCTAAGCAGCAGAGTGATGTTTG-3′by SUPERSCRIPT III One-Step RT-PCR Platinum Tag HiFi (Invitrogen)according to the following conditions: for RT; 55 C 30 min; 94 C, 2 min;for amplifying cDNA; 94 C, 30 s; 53 C, 30 s; 68 C, 1 min; ×40 cycles; 68C, 5 min. The 600 by human IL15 PCR-cDNA product was separated byelectrophoresis on a 1% agarose gel and isolated. The cDNA product waspurified from agarose with a Qiaquick Gel Extraction Kit (Qiagen). Thegene of the mature human IL15 protein was amplified from the 600 byhuman IL15 cDNA with the front primer

(SEQ ID NO: 21) 5′-TGGTTAACAACTGGGTGAATGTAATAAGTG-3′and the back primer

(SEQ ID NO: 22) 5′-ACGCGTTTATCAAGAAGTGTTGATGAACATTTGGAC-3′by PfuUltra (Stratagene) under following PCR conditions: 94 C, 1 min; 63C, 1 min; 72 C 1 min; ×35 cycles; 72 C, 10 min. The mature human IL15protein gene was gel-purified and cloned into the shuttle vector,pcDNA3.1 Directional TOPO Expression Vector (Invitrogen), with the TOPOreaction according to the manufacture's protocol. The clone containingthe mature human IL15 protein gene insert was identified based on thediagnostic PCR with the front primer

(SEQ ID NO: 21) 5′-TGGTTAACAACTGGGTGAATGTAATAAGTG-3′and the back primer

(SEQ ID NO: 22) 5′-ACGCGTTTATCAAGAAGTGTTGATGAACATTTGGAC-3′by RedTag (Sigma) under the following condition: 94 C, 1 min; 63 C, 1min; 72 C, 1 min; ×35 cycles; 72 C, 10 min. The sequence of the correctclone was verified by DNA sequencing with GenomeLab Dye TerminationCycle Sequencing with a QUICK START KIT (Beckman Coulter) according tothe manufacturer's protocol. The mature human IL15 protein gene wasremoved from the shuttle vector by digestion with HpaI and MluI andligated into an expression vector pNEF38-c264scTCR which had beendigested with HpaI and MluI. The pNEF38-c264scTCR expression vectorcontains the gene fragment encoding an immunoglobulin light chain leader(or secretory signal) sequence linked to the p53 (aa264-272)peptide-specific soluble chimeric single-chain TCR protein (c264scTCR)(Belmont, H. J. et al., 2006, Clin Immunol 121: 29-39). The vector alsocontains 5′ regulatory/enhancer regions, transcription regulatory andpromoter regions, translational regulatory/initiation/terminationsequences including a Kozak consensus sequence and poly-A terminationregion, and 3′ regulatory regions with putative matrix attachmentregulatory elements. The vector also contains DNA sequences allowingselective growth in mammalian cells (SV40 promoter/neoR gene/poly-A) andbacteria (ori/amp gene). Cloning of the DNA fragment encoding maturehuman IL15 protein into the pNEF38-c264scTCR vector resulted in ac264scTCR/huIL15 fusion gene comprising the following sequence: 3′-immunoglobulin light chain leader—264 TCR V-α—peptide linker—264 TCRV-β—human TCR C-β—human IL-15. The resulting vector(pNEF38-c264scTCR/huIL15), shown in FIG. 2A, was identified based on thediagnostic PCR and reconfirmed by DNA sequencing. The sequences of thec264scTCR/huIL15 fusion gene and protein (including the leader sequence)are shown at FIG. 2B and FIG. 2C, respectively.

Example 3 Construction of the c264scTCR/huIL15 Gene Fusion Containing a

Construction of the pNEF38-c264scTCR/huIL15 vector was described atExample 2. A mutated hinge region from human IgG1 H chain where threecysteine residues were substituted with three serine residues was usedto link c264scTCR and huIL15. The hinge region was mutated and amplifiedfrom 264scTCR/IgG1 gene described previously (Mosquera, L. A. et al.,2005, J Immunol 174: 4381-4388) with the front primer

(SEQ ID NO: 23) 5′-TGGTGGGTTAACGAGCCCAAATCTTCTG-3′and the back primer

(SEQ ID NO: 24) 5′-ATTATTACGCGTTGGAGACGGTGGAGATG-3′by PfuUltra (Stratagene) under following PCR conditions: 94 C, 30 sec;65 C, 30 sec; 70 C 1 min; ×35 cycles; 72 C, 10 min. The 70 by mutatedhuman IgG1 hinge PCR-cDNA product was separated by electrophoresis on a1% agarose gel and isolated. The cDNA product was purified from agarosewith a Qiaquick Gel Extraction Kit (Qiagen). The mutated hinge regiongene was digested with HpaI and MluI and ligated into pNEF38-c264scTCRwhich had been digested with HpaI and MluI. The clone containing themutated hinge region gene insert was identified based on the diagnosticPCR with the front primer

(SEQ ID NO: 25) 5′-TGAGTGATCGATACCACCATGGAGACAGACAC-3′and the back primer

(SEQ ID NO: 24) 5′-ATTATTACGCGTTGGAGACGGTGGAGATG-3′by RedTag (Sigma) under the following condition: 94 C, 30 sec; 64 C, 30sec; 70 C 1 min; ×35 cycles; 72 C, 10 min. The huIL15 was amplified frompNEF38-c264scTCR/huIL15 vector described at Example 2 with the frontprimer

(SEQ ID NO: 26) 5′-TGGTGGACGCGTAACTGGGTGAATG-3′and the back primer

(SEQ ID NO: 27) 5′-TGGTGGTCTAGAATTATCAAGAAGTGTTGATG-3′by PfuUltra (Stratagene) under following PCR conditions: 94 C, 30 sec;65 C, 30 sec; 70 C 1 min; ×35 cycles; 72 C, 10 min. The 380 by huIL15PCR-cDNA product was separated by electrophoresis on a 1% agarose geland isolated. The cDNA product was purified from agarose with a QiaquickGel Extraction Kit (Qiagen). The huIL15 gene was digested with MluI andXbaI and ligated into pNEF38-c264scTCR containing mutated hinge genewhich had been digested with MluI and XbaI. The clone containing thehuIL15 gene insert was identified based on the diagnostic PCR with thefront primer

(SEQ ID NO: 25) 5′-TGAGTGATCGATACCACCATGGAGACAGACAC-3′and the back primer

(SEQ ID NO: 27) 5′-TGGTGGTCTAGAATTATCAAGAAGTGTTGATG-3′by RedTag (Sigma) under the following condition: 94 C, 30 sec; 64 C, 2min; 70 C 2 min; ×35 cycles; 72 C, 10 min. The sequence of the correctclone was verified by DNA sequencing with GenomeLab Dye TerminationCycle Sequencing with a QUICK START KIT (Beckman Coulter) according tothe manufacturer's protocol. The pNEF38-c264scTCR expression vector isdescribed above at Example 2. Cloning of the DNA fragment encodingmutated human IgG1 hinge region and mature human IL15 protein into thepNEF38-c264scTCR vector resulted in a c264scTCR-hmt-huIL15 fusion genecomprising the following sequence: 3′-immunoglobulin light chainleader—264 TCR V-α—peptide linker—264 TCR V-β—human TCR C-β—mutatedhuman IgG1 hinge- human IL-15. The resulting vector(pNEF38-c264scTCR-hmt-huIL15), shown in FIG. 3A, was identified based onthe diagnostic PCR and reconfirmed by DNA sequencing. The sequences ofthe c264scTCR-hmt-huIL15 fusion gene and protein (including the leadersequence) are shown at FIG. 3B and FIG. 3C, respectively.

Example 4 Construction of the c264scTCR/huIL15RαΔE3 Gene Fusion in anExpression Vector

The total RNA of PBMC was prepared as described above. Human IL15Rα genecontaining coding region and a portion of 5′ and 3′ flanking regions wasamplified from the total RNA of the PBMC with the front primer

(SEQ ID NO: 28) 5′-AGTCCAGCGGTGTCCTGTGG-3′and the back primer

(SEQ ID NO: 29) 5′-TGACGCGTTTAAGTGGTGTCGCTGTGCCCTG-3′by SUPERSCRIPT III One-Step RT-PCR Platinum Tag HiFi (Invitrogen)according to the following condition: for RT; 55 C, 30 min; 94 C, 2 min;for amplifying cDNA; 94 C, 1 min; 66 C, 1 min; 72 C, 1 min; ×35 cycles;72 C, 5 min. The 970 by human IL15Rα PCR cDNA product was separated byelectrophoresis on a 1% agarose gel and isolated. The cDNA was purifiedfrom agarose with a Qiaquick Gel Extraction Kit (Qiagen). The humanIL15Rα extracellular domain gene was amplified from the 970 by humanIL15Rα cDNA with the front primer

(SEQ ID NO: 30) 5′-TGGTTAACATCACGTGCCCTCCCCCCATG-3′and the back primer

(SEQ ID NO: 29) 5′-TGACGCGTTTAAGTGGTGTCGCTGTGCCCTG-3′by PfuULTRA (Stratagene) under following PCR conditions: 94 C, 1 min; 72C 2 min; ×35 cycles, 72 C, 10 min. The human IL15Rα extracellular domaingene was gel-purified and cloned into the shuttle vector, pcDNA3.1Directional TOPO Expression Vector (Invitrogen), by TOPO reactionaccording to the manufacturer's protocol. The clone containing thecorrect human IL15Rα extracellular domain gene insert was chosen basedon diagnostic PCR and reconfirmed by DNA sequencing with the GenomeLabDye Termination Cycle Sequencing with a Quick Start Kit according to themanufacturer's protocol. The gene was determined to be human IL15RαΔE3extracellular domain gene. The human IL15RαΔE3 extracellular domain genewas removed from the shuttle vector by digestion with HpaI and MluI andligated into pNEF38-c264scTCR which had been digested with HpaI andMluI. Cloning of the DNA fragment encoding the human IL15RαΔE3extracellular domain into the pNEF38-c264scTCR vector resulted in ac264scTCR/huIL15Rα fusion gene comprising the following sequence:3′-immunoglobulin light chain leader—264 TCR V-α—peptide linker—264 TCRV-β—human TCR C-β—IL15RαΔE3 extracellular domain. The resulting vector(pNEF38-c264scTCR/huIL15RaDE3), shown in FIG. 4A, containing the correctIL15RαΔE3 extracellular domain gene insert was identified based on thediagnostic PCR and reconfirmed by DNA sequencing. The sequences of thec264scTCR/huIL15RαΔE3 gene and protein are shown at FIG. 4B and FIG. 4C,respectively.

Example 5 Construction of c264scTCR/huIL15RαSushi Gene Fusion in anExpression Vector

The total RNA of PBMC was prepared as described above. Human IL15RαSushigene was amplified from the 970 by human IL15Rα cDNA (see Example 3)with the front primer

(SEQ ID NO: 30) 5′-TGGTTAACATCACGTGCCCTCCCCCCATG-3′and the back primer

(SEQ ID NO: 31) 5′-TTGTTGACGCGTTTATCTAATGCATTTGAGACTGG-3′by PfuULTRA (Stratagene) under following PCR conditions: 94 C, 1 min; 66C, 1 min; 70 C, 1 min; ×35 cycles; 72 C, 10 min. The PCR product ofhuman IL15RαSushi gene was gel-purified and digested with HpaI and MluI.The gene was ligated into pNEF38-c264scTCR which had been digested withHpaI and MluI. Cloning of the DNA fragment encoding the humanIL15R□Sushi domain into the pNEF38-c264scTCR vector resulted in ac264scTCR/huIL15Rα fusion gene comprising the following sequence:3′-immunoglobulin light chain leader—264 TCR V-α—peptide linker—264 TCRV-β—human TCR C-β—human IL15RαSushi. The resulting vector, shown in FIG.5A, containing the correct human IL15RαSushi gene insert was identifiedbased on the diagnostic PCR and reconfirmed by DNA sequencing. Thesequences of the c264scTCR/huIL15RαSushi gene and protein are shown atFIG. 5B and FIG. 5C, respectively.

Example 6 Construction of c264scTCR/huIL15RαSushi Gene Fusion Containinga Mutated Human IgG1 Hinge Region in an Expression Vector

Construction of the pNEF38-c264scTCR/huIL15RαSushi vector was describedabove. A mutated hinge region from human IgG1 H chain where threecysteine residues were replaced by three serine residues was used tolink c264scTCR and huIL15RαSushi. The hinge region was mutated,amplified, ligated, and verified as above. The huIL15RαSushi wasamplified from pNEF38-c264scTCR/huIL15RαSushi vector described abovewith the front primer

(SEQ ID NO: 32) 5′-TAATAAACGCGTATCACGTGCCCTC-3′and the back primer

(SEQ ID NO: 33) 5′-TGGTGGTCTAGATTATCATCTAATGCATTTG-3′by PfuUltra (Stratagene) under following PCR conditions: 94 C, 30 sec;65 C, 30 sec; 70 C 1 min; ×35 cycles; 72 C, 10 min. The 250 byhuIL15RαSushi PCR-cDNA product was separated by electrophoresis on a 1%agarose gel and isolated. The cDNA product was purified from agarosewith a Qiaquick Gel Extraction Kit (Qiagen). The huIL15RαSushi gene wasdigested with MluI and XbaI and ligated into pNEF38-c264scTCR containingmutated hinge gene which had been digested with MluI and XbaI. The clonecontaining the huIL15 gene insert was identified based on the diagnosticPCR with the front primer

(SEQ ID NO: 23) 5′-TGGTGGGTTAACGAGCCCAAATCTTCTG-3′and the back primer

(SEQ ID NO: 33) 5′-TGGTGGTCTAGATTATCATCTAATGCATTTG-3′by RedTag (Sigma) under the following condition: 94 C, 30 sec; 65 C, 1min; 70 C 1 min; ×35 cycles; 72 C, 10 min. The sequence of the correctclone was verified by DNA sequencing with GenomeLab Dye TerminationCycle Sequencing with a QUICK START KIT (Beckman Coulter) according tothe manufacturer's protocol. The pNEF38-c264scTCR expression vector isdescribed above. Cloning of the DNA fragment encoding mutated human IgG1hinge region and human IL15RαSushi protein into the pNEF38-c264scTCRvector resulted in a c264scTCR-hmt-huIL15RαSushi fusion gene comprisingthe following sequence: 3′- immunoglobulin light chain leader—264 TCRV-α—peptide linker—264 TCR V-β—human TCR C-β—mutated human IgG1 hinge-human IL15RαSushi. The resulting vector(pNEF38-c264scTCR-hmt-huIL15RαSushi), shown in FIG. 6A, was identifiedbased on the diagnostic PCR and reconfirmed by DNA sequencing. Thesequences of the c264scTCR-hmt-huIL15R□Sushi fusion gene and protein(including the leader sequence) are shown at FIG. 6B and FIG. 6C,respectively.

Example 7 Construction of the c264scTCR/huIL15RαSushi andc264scTCR/huIL15 Genes in a Single Expression Vector

To achieved expression of two fusion proteins of the invention in asingle host cell, the genes encoding c264scTCR/huIL15RαSushi andc264scTCR/huIL15 were cloned into a single expression vector. Thec264scTCR/huIL15RαSushi gene was amplified from the template describedin Example 5 by PfuUltra (Stratagene) with the front primer5′-TGAGTGTCCGGAACCACCATGGAGACAGACAC-3′ (SEQ ID NO: 34) and the backprimer 5′-TTGTTGGCGGCCGCTTATCATCTAATGCATTTGAG-3′ (SEQ ID NO: 35) underthe following condition: 94 C, 1 min; 68C, 1 min; 72 C, 2 min; ×35cycles; 72 C, 10 min. The PCR product of c264scTCR/huIL15RαSushi genewas gel-purified, digested with BspEI and NotI and ligated into thepSUN34R1 expression vector which had been digested with BspEI and NotI.The pSUN34R1 expression vector contains two sites for cloninggenes-of-interest as well as 5′ regulatory/enhancer regions,transcription regulatory and promoter regions, translationalregulatory/initiation/termination sequences including a Kozak consensussequence and poly-A termination region, and intron and 3′ regions withregulatory elements. This vector also contains DNA sequences allowingselective growth in mammalian cells (SV40 promoter/neoR gene/poly-A) andbacteria (ori/amp gene). The vector containing the correctc264scTCR/IL15RαSushi gene insert was identified based on the diagnosticPCR and reconfirmed by DNA sequencing. The c264scTCR/huIL15 gene wasamplified from the template described in Example 2 by PfuUltra(Stratagene) with the front primer

(SEQ ID NO: 25) 5′-TGAGTGATCGATACCACCATGGAGACAGACAC-3′and the back primer

(SEQ ID NO: 36) 5′-TGAGTGTTCGAATTATCAAGAAGTGTTGATGAAC-3′under the following condition: 94 C, 1 min; 65 C, 1 min; 72 C, 2 min;×35 cycles; 72 C, 10 min. The PCR product of c264scTCR/huIL15 gene wasgel-purified, digested with ClaI and Csp45I and ligated intopSUN34R1-c264scTCR/huIL15RαSushi expression vector which had beendigested with ClaI and Csp45I. The resulting vector(pSun-c264scTCRIL15/c264scTCRIL15RaSushi), shown in FIG. 7, containingthe correct c264scTCR/huIL15 gene insert was identified based on thediagnostic PCR and reconfirmed by DNA sequencing. This vector containsboth c264scTCR/huIL15RαSushi and c264scTCR/huIL15 genes.

Example 8 Construction of c264scTCR/huIL15RαΔE3 and c264scTCR/huIL15Genes in a Single Expression Vector

The c264scTCR/huIL15R⋆ΔE3 fusion gene was amplified from the templatedescribed in Example 4 by PfuUltra (Stratagene) with the front primer

(SEQ ID NO: 34) 5′-TGAGTGTCCGGAACCACCATGGAGACAGACAC-3′and the back primer

(SEQ ID NO: 37) 5′-TTGTTGGCGGCCGCTTATCAAGTGGTGTCGCTG-3′under the following condition: 94 C, 1 min; 68 C, 1 min; 72 C, 2 min;×35 cycles; 72 C, 10 min. The PCR product of c264scTCR/huIL15RαΔE3 genewas gel-purified, digested with BspEI and NotI and ligated to theexpression vector pSUN34R1 which had been digested with BspEI and NotI.The vector containing the correct c264scTCR/huIL15RαΔE3 gene insert wasidentified based on the diagnostic PCR and reconfirmed by DNAsequencing. The c264scTCR/huIL15 gene was amplified and cloned into theexpression vector as described on Example 7. The resulting vector(pSun-c264scTCRIL15/c264scTCRIL15RaDE3), shown in FIG. 8, containing thecorrect c264scTCR/huIL15 gene insert was identified based on thediagnostic PCR and reconfirmed by DNA sequencing. This vector containsboth c264scTCR/huIL15RαΔE3 and c264scTCR/huIL15 genes.

Example 9 Generation of Transfected Host Cell Lines Producing FusionProteins

The expression vectors can be introduced into a variety of host celllines by several different transformation, transfection or transductionmethods. In one such method, CHO-K1 cells (5×10⁴) were seeded in a6-well plate and cultured overnight in a CO₂ incubator. The cells weretransfected with 5 μg of expression vector containing the TCR/IL15and/or TCR/IL15Rα fusion genes using 10 μL of Mirus TransIT-LT1 reagent(Mirus) according to the manufacturer's protocol. The cells wereselected with 4 mg/mL of G418 (Invitrogen) one day after thetransfection. The G418 resistant cells were expanded and TCR fusionprotein expressing cells were enriched by 3-5 rounds of MACS selectionas described below. The cells were detached in 10 mM EDTA and washedonce with IMDM containing 10% FBS. Cells were resuspended (10⁷ cells in100 μL) and incubated with 5 μg of R-Phycoerythrin (PE) conjugated p53(aa264-272)/HLA-A2 tetramer reagent for 15 min at 4 C. The cells werewashed once and incubated with anti-PE antibody conjugated magneticbeads (Miltenyi Biotec) for 15 min at 4 C. The cells were loaded to amagnetic column (in a magnetic field) and the unbound cells were removedwith wash buffer (PBS containing 0.5% BSA). The column-bound cells wereeluted with IMDM containing 10% FBS after the column had been removedfrom the magnetic field. This procedure allows enrichment of fusionprotein-expressing cells based on the transient display of the solublefusion protein on the cell surface during the production/secretionprocess. The cell surface association of the fusion proteins wasmonitored after each enrichment. Levels of cell surface-bound fusionproteins determined by flow cytometry were compared to levels of solublefusion proteins present in the cell culture media as determined byELISA. An example of the comparison is shown in FIGS. 9A and 9B. In thisexample, CHO-K1 cells transfected with pNEF38-c264scTCR/huIL15RαSushiwere enriched by MACS for one to five times and were then seeded (1×10⁶cells/well) on a 6-well plate. After 24 hours, cells were then detachedwith 10 mM EDTA, washed once with IMDM+10% FBS, and stained (at 2×10⁵cells/100 μL of IMDM+10% FBS) with 0.6 μg of PE-conjugated p53(aa264-272)/HLA-A2 tetramer or same amount of control PE-conjugatedCMVpp65 (aa495-503)/HLA-A2 tetramer for 30 min at 4 C. Cells were washedonce and analyzed for levels of cell surface associated soluble fusionprotein by flow cytometry, as shown in FIG. 9A. The level of solublefusion protein secreted into the cell culture medium was also determinedby TCR-specific ELISA with a capture antibody, anti-human TCR Cβantibody (BF1), and a detection antibody, biotinylated anti-human TCR Cβantibody (W4F) described previously (5), as shown in FIG. 9B. Theresults indicate that the magnetic bead-based enrichment process yieldedtransfectants that produced increased levels of soluble fusion protein.The enriched transfected cells were then subcloned three times by thelimiting dilution and production cell lines were screened based on thelevel of soluble fusion protein secreted into the culture media(determined by ELISA described above). Production cell lines wereexpanded and grown in IMDM+10% FBS or serum-free media under conditions(i.e. flasks, spinners, fermenters, bags, bottles) suitable to generatethe soluble fusion protein.

In some cases, host cells were co-transfected with different expressionvectors to generate transfectants capable of expressing multiple fusionproteins. Transfectants expressing one fusion protein could also bere-transfected with a one or more expression vectors to generatetransfectants expressing multiple fusion proteins. Cells were alsotransfected with an expression vector containing more that one fusionprotein genes, as exemplified in Examples 7 and 8, to generate atransfectant expressing multiple fusion proteins. The resulting cellscould be used to produce the multi-component fusion protein complexes ofthe invention as soluble molecules in the cell culture medium.

High levels of fusion protein or fusion protein complex production canalso be achieved through cell transfection and selection methodsdescribed in U.S. Ser. No. 09/204,979.

Example 10 Purification of the TCR/IL15 and TCR/IL15Rα Fusion Proteinsor Fusion Protein Complexes

Soluble fusion proteins or fusion protein complexes of the invention canbe purified from the host cells or cell culture media using a variety ofmethods, including by selective partitioning or solubility in solventsor by separation (i.e. via chromatography) based on charge,hydrophobicity, hydrophilicity, size, and/or selective or semi-selectivebinding to a ligand. Soluble fusion proteins or fusion protein complexescan be generated from insoluble materials through use of the appropriateprotein folding conditions. In one example, c264scTCR/IL15 fusionprotein was purified from cell culture media by affinity chromatographyusing a antibody (BF1) recognizing the human TCR-Cβ domain. Typically, acolumn containing BF1-conjugated Sepharose was first equilibrated with20 mM Tris-HCl pH 8.0 (loading buffer) and then loaded at 2 ml/min withpH adjusted cell culture media containing c264scTCR/IL15 fusion protein.The column was then washed with 5 column volumes of the loading bufferto remove unbound proteins, and the c264scTCR/IL15 fusion protein waseluted with 4 column volumes of 0.5M Na-citrate, pH 4. After collection,the eluate was adjusted to pH 8.0 by 2M Tris-HCl pH 8.0. The purifiedprotein was buffer exchanged into PBS and filtered using 0.22 μm filter.The BF1 column was stripped with 50 mM Glycine-HCl pH 3.0, and stored in20% ethanol at 4 C for further use. The fusion protein could be furtherpurified by ion exchange and/or size exclusion chromatography. Cellculture supernatants containing c264scTCR/IL15, c264scTCR/IL15RαSushiand c264scTCR/IL15RαΔE3 fusion proteins were purified by the abovemethods and samples of the purified fusion proteins were analyzed byelectorphoresis on SDS polyacrylamide gels under reducing conditions andfollowed by staining with Coomassie brilliant blue. Examples of suchgels are shown in FIG. 10. The major protein bands correspond to thecorrect molecular weights expected based on fusion protein sequences.

Example 11 Generation of a Fusion Protein Complex of the TCR/IL15 andTCR/IL15Rα Fusion Proteins

IL15 specifically binds to the extracellular IL15Rα domain with highaffinity (4). Thus a complex of fusion proteins bearing the IL-15 andIL15Rα domains can be formed under a variety of conditions, includingwithin the expression cell or extracellularly with unpurified orpurified fusion proteins. In one example, equal molar amounts ofpurified fusion proteins can be mixed under the appropriate conditions(i.e. 10 min at room temperature) to form a fusion protein complex.Complex formation can be monitored in using a variety of techniquesincluding direct binding assays, competitive binding assays,immunoprecipitation, surface plasma resonance, or analyses based oncomplex size, activity or other properties. For example, as shown inFIG. 11, size exclusion chromatography can monitor the formation ofcomplexes comprising c264scTCR/huIL15 and c264scTCR/huIL15RαSushi fusionproteins based on molecular weight. In this study, about 100 μg ofc264scTCR/huIL15 (0.5 mg/ml) was loaded on a Superdex 200 HR 10/30column for the analysis. The calculated molecular weight forc264scTCR/huIL15 is about 57 kD. Based on SEC profile (FIG. 11A), theestimated molecular weight is about 98 kD, suggesting that this fusionprotein is likely a monomer. Similarly, about 60 μg ofc264scTCR/huIL15RαSushi fusion protein (0.3 mg/ml) was loaded on theSuperdex column. The calculated molecular weight forc264scTCR/huIL15RαSushi is about 52 kD. Based on SEC profile (FIG. 11B),estimated molecular weight of the fusion protein is about 81 kD, againsuggesting this fusion protein is a monomer. Previous SEC analysis ofother TCR-based fusion proteins showed similar differences between thecalculated monomeric molecular weight and the estimated molecular weightof the glycosylated fusion protein. When the c264scTCR/huIL15 andc264scTCR/huIL15RαSushi fusion proteins were mixed in equal molaramounts and about 126 μg of the mixed proteins (0.63 mg/ml) were loadedon the column, the profile shown in FIG. 11C was obtained. Molecularweights of two major peaks were estimated: one at about 170 kD, which isa heterodimer of the two fusion proteins and another one at about 91 kD,which is likely a mix of monomeric forms of the fusion proteins. Thus,the appearance of the 170 kD species in the mixedc264scTCR/huIL15+c264scTCR/huIL15RαSushi fusion protein preparation isevidence that the fusion protein complex of the invention can begenerated.

Analysis of the fusion protein complex comprising c264scTCR/huIL15 andc264scTCR/huIL15RαΔE3 fusion proteins was also conducted. About 100 μgof c264scTCR/huIL15RαΔE3 fusion protein (0.5 mg/ml) was loaded on theSuperdex column. The calculated molecular weight forc264scTCR/huIL15RαΔE3 is about 60 kD. Based on SEC profile (FIG. 12A),estimated molecular weight of the protein is about 173 KD, suggestingthis protein exists as a homodimer. When the c264scTCR/huIL15 andc264scTCR/huIL15RαΔE3 fusion proteins were mixed in equal molar amountsand about 118 μg of the mixed proteins (0.59 mg/ml) were loaded on thecolumn, the profile shown in FIG. 12B was obtained. Molecular weights oftwo major peaks were estimated: one is >210 kD, which is likely atetramer composed of two heterodimers and the other is about 93 kD,likely to be c264scTCR/huIL15 monomer. Thus, the appearance of the 170kD species in the mixed c264scTCR/huIL15+c264scTCR/huIL15RαΔE3 fusionprotein preparation is evidence that the fusion protein complex of theinvention can be generated.

Example 12 Fusion Protein Complex of the TCR/IL15 and TCR/IL15Rα FusionProteins Exhibits Enhanced Binding for Peptide/MHC Complexes

The fusion protein complexes generated as described above werecharacterized for their ability to bind the TCR-specific antigen, p53(aa264-272)/HLA-A2.1. To generate cells presenting this antigen,HLA-A2.1-positive T2 cells were loaded with p53 (aa264-272) peptide at26 C overnight and then stored at 5×10⁶ cells/mL in a liquid nitrogen.T2 cells that were not incubated with peptide serve as controls. The p53peptide-loaded or control T2 cells were thawed and resuspended in 1 mLof IMDM+10% FBS. The cells (5×10⁵/100 μL) were then stained for 30 minat RT with 0.5 μg of following fusion proteins: c264scTCR/huIL15,c264scTCR/huIL15RαSushi, c264scTCR/huIL15+c264scTCR/huIL15RαSushicomplex. Cells were washed once with washing buffer (PBS containing 0.5%BSA and 0.05% sodium azide) and stained with 0.1 μg of biotinylatedmouse monoclonal anti-human TCR Cβ antibody (BF1) in 100 μL of washingbuffer for 30 min at RT. Cells were washed once and stained with 0.5 μgof R-Phycoerythrin conjugated streptavidin in 100 μL of washing bufferfor 30 min at RT. Cells were washed and resuspended for analysis by flowcytometry. As shown in FIG. 13, each of the fusion proteins was capableof specifically staining p53 peptide-loaded cells. In addition, thec264scTCR/huIL15+c264scTCR/huIL15RαSushi fusion protein complexdisplayed enhanced specific binding via the multivalent c264scTCRdomains to the p53 (aa264-272)/HLA-A2.1 complexes displayed on the T2cells. In particular, the dimeric fusion protein complex showed betterstaining of the p53 peptide-loaded T2 cells than the monomericc264scTCR/huIL15 or c264scTCR/huIL15RαSushi fusion proteins. These datasuggest that the multimeric fusion protein complex will provide betterantigen recognition properties than monomeric form of the fusionproteins.

Example 13 Generation of huIL-15 Mutant Genes and Construction ofc264scTCR-hmt-huIL15 Mutant Gene Expression Vectors

As described above, c264scTCR/huIL15+c264scTCR/huIL15RαSushipolypeptides are able to form a complex through interactions of theIL-15 and IL-15Rα domains and the multivalent fusion protein complex hasenhanced binding for peptide/MHC complexes. Such a fusion proteincomplex has advantages as an antigen-specific or targeted research,diagnostic and therapeutic agent based on the enhanced binding activity.The ability of the IL-15/IL-15Rα domains of the fusion protein to bindcells expressing IL-15 receptors is also an desirable feature asindicated herein. However, there are applications where it isadvantageous to increase or decrease the ability of the IL-15/IL-15Rαdomains to interact with and/or effect the responses of cells expressingIL15 receptors. For example; it may be desirable to reduce thisinteraction in the applications (i.e. research and diagnostic uses)where the primary goal is to use the fusion protein complex for specificdetection of peptide/MHC complexes. In therapeutic applications, it mayalso be desirable to generated fusion protein complexes that containIL-15 domains capable of increasing or decreasing IL-15-mediatedresponses. To address this issue, mutational analysis was carried out toidentify residues in IL-15 that effect its binding to IL-2/15Rβγ_(C)complex without effecting its interactions to IL-15Rα. The resultingmutations may create IL-15 variants including antagonists or agonists.In addition to use in the fusion proteins of the invention, theresulting IL-15 antagonists and agonists also may have utility assoluble cytokines (i.e., non-fusion proteins) or as a complex withIL-15Rα domains, for research, diagnostic or therapeutic applications.For example, IL-15 antagonists may be useful in suppressing unwantedimmune responses whereas IL-15 agonists may be used to stimulate immuneresponses in therapeutic strategies to treat various diseases.

Based on a comparison between the amino acid sequence and structure ofIL-15 with IL-2, several amino acids were identified that couldpotentially effect interactions between IL-15 and IL-15Rα, IL-15Rβand/or γC. As showed in Table 1 and FIG. 14A, IL-15 variants werecreated where the potential binding sites of the mature human IL-15protein to IL-15Rβγ_(C) receptors, amino acids at positions 8, 61, 65,72, and 108 (numbering based on mature native human IL-15 sequence),were each substituted or combined with two or more other substitutions.The aspartic acid at position 8 was substituted with alanine orasparagine. The aspartic acid at position 61 was substituted withalanine. The asparagine at position 65 was substituted with alanine oraspartic acid. The asparagine at position 72 was substituted witharginine or aspartic acid. The glutamine at position 108 was substitutedwith alanine. Both Asp at position 8 and Gln at position 108 were eachsubstituted with an alanine. Both Asp at position 8 and Asn at position65 were each substituted with an asparagine or alanine. Both Asp atposition 8 and Asn at position 65 were each substituted with a serine orarginine. To generate IL-15 mutants, the overlapping PCR was used.

For example, to generate Asp at position 8 with the substitution ofalanine or asparagines residues, pNEF38-c264scTCR/huIL15 vector was usedas template to amplify two overlapping c-DNA fragments with the frontprimer for fragment 1

(SEQ ID NO: 26) 5′-TGGTGGACGCGTAACTGGGTGAATG-3′and with the back primer for fragment 1

(SEQ ID NO: 38) 5′-AGATCTTCAATTTTTTTCAAMKHACTTATTACATTCACCCAG-3′and with the front primer for fragment 2

(SEQ ID NO: 39) 5′-ACTGGGTGAATGTAATAAGTDMKTTGAAAAAAATTGAAGATC-3′and with the back primer for fragment 2

(SEQ ID NO: 40) 5′-TGGTGGTCTAGATTATCAAGAAGTGTTGATG-3′by PfuUltra (Stratagene) under following PCR conditions: 94 C, 1 min; 66C, 1.5 min; 72 C 1.5 min; ×35 cycles; 72 C, 10 min. The fragments 1 and2 PCR-cDNA products were separated by electrophoresis on a 1% agarosegel and isolated. The cDNA product was purified from agarose with aQiaquick Gel Extraction Kit (Qiagen). The fragments 1 and 2 PCR-cDNAproducts were fused together with PfuUltra (Stratagene) under followingPCR conditions: 94 C, 1 min; 66 C, 1.5 min; 72 C 1.5 min; ×10 cycles.The overlapping PCR-cDNA fragment was amplified with the front primer

(SEQ ID NO: 26) 5′-TGGTGGACGCGTAACTGGGTGAATG-3′and with the back primer

(SEQ ID NO: 40) 5′-TGGTGGTCTAGATTATCAAGAAGTGTTGATG-3′by PfuUltra (Stratagene) under following PCR conditions: 94 C, 1 min; 64C, 1.5 min; 69 C 1.5 min; ×30 cycles; 72 C, 10 min. The huIL-15 mutantPCR-cDNA products were separated by electrophoresis on a 1% agarose geland isolated. The cDNA product was purified from agarose with a QiaquickGel Extraction Kit (Qiagen). The huIL15 mutant gene was digested withMluI and XbaI and ligated into pNEF38-c264scTCR-hmt which had beendigested with MluI and XbaI. The clone containing the huIL15 gene atposition 8 with a substitution of alanine or asparagine was verified byDNA sequencing with GenomeLab Dye Termination Cycle Sequencing with aQUICK START KIT (Beckman Coulter) according to the manufacturer'sprotocol. The pNEF38-c264scTCR expression vector is described above.Cloning of the DNA fragment encoding mutated human IL-15 protein intothe pNEF38-c264scTCR vector resulted in a c264scTCR-hmt-huIL15D8A orc264scTCR-hmt-huIL15D8N fusion gene comprising the following sequence:3′-immunoglobulin light chain leader—264 TCR V-α—peptide linker—264 TCRV-β—human TCR C-β—mutated human IgG1 hinge-human IL15D8A or —humanIL15D8N. The resulting vector (pNEF38-c264scTCR-hmt-huIL15D8A orpNEF38-c264scTCR-hmt-huIL15D8N), shown in FIG. 14B, was confirmed by DNAsequencing. The sequences of the pNEF38-c264scTCR-hmt-huIL15D8A orpNEF38-c264scTCR-hmt-huIL15D8N fusion gene and protein (including theleader sequence) are shown at FIG. 14C and FIG. 14D, respectively.

Other mutations were introduced in a similar fashion and expressionvector constructed as described above. The expression vectors wereintroduced into CHO.K1 cells to generated stable transfectants asdescribed in Example 9. Production and purification of the TCR/IL15fusion proteins and fusion protein complexes comprising IL-15 variantswas carried out using similar methods as described in Examples 10 and11. Generation of IL-15 variants as soluble cytokines can be carried outthrough a variety of methods known in the art, including production inprokaryotic and eukaryotic expression systems (see for example,WO9527722; Hsu et al. 2005 J. Immunol. 175:7226).

Example 14 Functional Characterization of the TCR/IL15 and TCR/IL15RαFusion Proteins and Fusion Protein Complexes

Functional binding of the fusion protein TCR domain was demonstratedbased on the ELISA and cell staining methods using p53(aa264-272)/HLA-A2.1 reagents described in Example 9 and the antigenpresenting cell staining methods described in Example 12. The ability ofthe fusion protein IL15 and IL15Rα domains to interact was demonstratedas described in Example 11. Further the functional activity of the IL15and IL15Rα domains can be assessed through a variety of methodsincluding binding to IL-2/15Rβγ_(C) receptors or modulation of theactivity of immune cells bearing IL-15 receptors. In one example, CTLL-2cells, which express the heterotrimeric IL-15R (αβγ_(c) chains), wereincubated with 0.5 μg of the individual fusion proteins:c264scTCR/huIL15, 264scTCR/huIL15RαSushi, orc264scTCR/huIL15+c264scTCR/huIL15RαSushi complex for 30 min at RT. Cellswere washed once with washing buffer (PBS containing 0.5% BSA and 0.05%sodium azide) and stained with 0.5 μg of R-Phycoerythrin (PE) conjugatedp53 (aa264-272)/HLA-A2 tetramer for 30 min at RT. Cells were washed andresuspended for analysis by flow cytometry. As shown in FIG. 15,association of the c264scTCR/huIL15 fusion protein andc264scTCR/huIL15+c264scTCR/huIL15RαSushi complex via their huIL15domains with the IL-15 receptors on CTLL-2 cells can be detected withPE-conjugated p53 (aa264-272)/HLA-A2 tetramer recognizing the c264scTCRdomain of the bound fusion protein. These results indicate that both theIL-15 and TCR domains of the fusion protein/fusion protein complexes arecapable of functionally interacting with their cognate ligands.

In addition, CTLL-2 cells are dependent upon cytokines for growth andcan respond to recombinant human IL-15. A cell-based WST-1 proliferationassay using CTLL-2 cells was developed to assess the IL-15 biologicalactivity of fusion proteins and fusion protein complexes. WST-1 (Roche)is a reagent that can be converted into formazan by dehydrogenaseenzymes found in metabolically active cells. In the WST-1 assay, thequantity of formazan in the culture media measured by the amount of440-450 nm absorbance is directly proportional to the number of livingcells in culture. CTLL-2 cells (2×10⁴/200 μL) were incubated with theindicated concentrations of fusion proteins (0-28 ng/mL):c264scTCR/huIL15, c264scTCR/huIL15+c264scTCR/huIL15RαSushi complex orc264scTCR/huIL15+c264scTCR/huIL15RαΔE3 complex for 3 days in 96-wellplates at 37 C in a CO₂ incubator. Cells were incubated with 10 μL ofWST-1 for 4 hours before harvesting 100 μL of culture medium for 440-450nm absorbance measurement with a microtiter plate reader. As shown inFIG. 16, c264scTCR/huIL15 fusion protein can support the proliferationof CTLL-2 cells at a concentration as low as 1.8 ng/mL (˜31.25 pM),suggesting activation of CTLL-2 cells with c264scTCR/huIL15 fusionprotein via the high affinity IL15 receptor. Interestingly, fusionprotein complexes also supported CTLL-2 cell proliferation but to alesser degree suggesting that c264scTCR/huIL15 stimulatory activity wasinhibited following complex formation with c264scTCR/huIL15RαSushi orc264scTCR/huIL15RαΔE3 (by one fold or four fold, respectively). Thissuggests the binding of c264scTCR/huIL15 to the high affinity IL15receptor is inhibited by c264scTCR/huIL15RαSushi orc264scTCR/huIL15RαΔE3 fusion proteins. These results provide evidencethat the fusion proteins and fusion protein complexes can activate orsuppress responses of immune cells under different conditions.

Similar assays were performed with cell lines expressing only theintermediate affinity IL-15βγ_(c) receptors, such as 32Dβ cell lines(see below). In some cases, it is possible that the biological activityof IL15 in stimulating proliferation of theIL-15R-bearing cells will beenhanced when it is in a complex with the IL15Rα domain (Mortier, E. etal., 2006, J. Biol. Chem.,281: 1612-1619; Stoklasek, T. et al., 2006, JImmunol 177: 6072-6080; Rubinstein, M. P. et al., 2006, Proc Natl AcadSci U S A 103: 9166-9171). Stimulation of cell proliferation by thec264scTCR/huIL15+c264scTCR/huIL15RαSushi orc264scTCR/huIL15+c264scTCR/huIL15RαΔE3 complexes will be assessed andmay provide additional evidence that the fusion protein complexes canstimulate or activate immune responses of immune cells.

Example 15 Dimeric Fusion Protein Complexes of the TCR/IL15RαSushi andTCR/IL15 Variants Exhibit TCR-Specific Binding to Peptide/MHC Complexbut Less Binding to the IL-15Rβγ_(C) Receptors

The fusion protein complexes comprising IL-15 variants as describedabove were characterized for their ability to bind the TCR-specificantigen, p53 (aa264-272)/HLA-A2.1. To generate cells presenting p53(aa264-272)/HLA-A2.1, HLA-A2.1-positive T2 cells (2×10⁶/mL) were loadedwith 20 μM p53 (aa264-272) peptide at 37 C in the presence of 1×PLE(Altor Bioscience) for 2-3 hrs. T2 cells that were not incubated withpeptide and 32Dβ cells expressing IL-2/15Rβγ_(C) serve as controls. Thep53 peptide-loaded T2 cells, control T2 cells, or 32Dβ cells (2×10⁵/100μL) were then incubated for 30 min at 4 C with 320 nM of followingdimeric fusion protein complexes: 1)c264scTCR/huIL15+c264scTCR/huIL15RαSushi, 2)c264scTCR/huIL15D8A+c264scTCR/huIL15RαSushi, and 3)c264scTCR/huIL15D8N+c264scTCR/huIL15RαSushi. These complexes weregenerated by incubating 160 nM of purified c264scTCRhuIL15 fusionprotein and 160 nM of purified c264scTCRhuIL15RαSushi fusion protein at4 C for 3 hours: Following staining, cells were washed once with washingbuffer (PBS containing 0.5% BSA and 0.05% sodium azide) and stained with0.5 μg of biotinylated mouse monoclonal anti-human TCR Cβ antibody (BF1)in 100 μL of washing buffer for 30 min at 4 C. Cells were washed onceand stained with 0.5 μg of R-Phycoerythrin conjugated streptavidin in100 μL of washing buffer for 30 min at 4 C. Cells were washed andresuspended for analysis by flow cytometry. As shown in FIG. 17A, thec264scTCR/huIL15D8A+c264scTCR/huIL15RαSushi complex andc264scTCR/huIL15D8N+c264scTCR/huIL15RαSushi complex exhibited equivalentactivity as the c264scTCR/huIL15+c264scTCR/huIL15RαSushi complex forspecifically staining p53 peptide-loaded T2 cells. These resultsindicate that the multivalent scTCR domains are fully functional in eachof these fusion complexes. However, as shown in FIG. 17B and FIG. 17C,the mutant c264scTCR/huIL15 fusion protein complexes showed lessbackground staining on control T2 cells (FIG. 17B) andIL-15Rβγ_(c)—positive 32Dβ cells (FIG. 17C) than the wide typec264scTCR/IL15 fusion protein complex. Thus these fusion proteincomplexes comprising IL-15 variants (D8A and D8N) do not show bindingactivity to the IL-15Rβγ_(c) receptors present on the 32Dβ cells.Similar studies of IL-15Rβγ_(c) receptor binding were carried out withother fusion proteins comprising IL-15 variants and are summarized inTable 1. The results indicate that fusion proteins and fusion proteincomplexes of the invention comprising IL-15 variants retain activity torecognize peptide/MHC complexes and exhibit decreased or increasedbinding activity for IL-15Rβγ_(c) receptors.

To confirm the above fusion proteins functional TCR and IL-15 domains,peptide/MHC and IL-15Ra binding activity was measured by ELISA analysis.The 96-well microtiter plates were precoated with 20 nM BF1, an anti-TCRCβ antibody, or 20 nM TCR/IL15RαSushi in carbonated buffer pH 9.1(sodium bicarbonate 35 mM, Na₂CO₃, 17.5 mM, NaCl 50 mM) over 3 hours at4 C. Plates were washed with washing buffer (Imidazole 40 mM, NaCl 150mM) for 4 times and blocked with 1% BSA-PBS for 10 minutes. Theindicated fusion proteins at a concentration of 0.03-4 nM were added tothe plates and incubated at RT for 30 minutes. The plates were washedfour times. The BF1 captured fusion proteins were incubated with 1 μg/mLof HRP-conjugated p53/HLA-A2.1 tetramer for 45 minutes at RT and theTCR/IL15RaSushi captured fusion proteins were incubated with 50 ng/mL ofbiotinylate mouse anti-human IL-15 for 30 minutes at RT. After washingfor 4 times, the plate incubated with biotinylate mouse anti-human IL-15was incubated with 0.25 μg/mL of HRP-streptavidin for 15 minutes. Theplates were washed 4 times and incubated with peroxidase substrate ABTfor 1-10 minutes and developed for 405 nm absorbance measurement with amicrotiter plate reader. As shown at FIG. 18A and FIG. 18B, the fusionproteins shared similar TCR-specific binding activity for p53/HLA-A2tetramer and equivalent IL-15 binding activity for IL15RαSushi. Similarstudies of IL-15Rα binding were carried out with other fusion proteinscomprising IL-15 variants and are summarized in Table 1. The resultsindicate that fusion proteins and fusion protein complexes of theinvention comprising IL-15 variants retain activity to recognizepeptide/MHC complexes and IL-15Rα receptors.

Example 16 Functional Characterization of the TCR/IL15 Mutant FusionProteins and Fusion Protein Complexes

As indicated above, fusion proteins comprising an IL-15 antagonist oragonist may be a useful as a targeted agents for inhibiting orstimulating IL-15-mediated responses (i.e., T cell or NK cell activity)at the disease site. To determine the IL-15 bioactivity of these fusionproteins to effect immune responses, cell proliferation studies werecarried out with CTLL-2 cells expressing the high affinity IL-15R(αβγ_(c) chains) and with 32Dβ cells expressing the intermediate IL-15R(βγ_(c) chains). The cells (2×10⁴/200 μL) were incubated with 0.4-40 nMof the above described TCR/IL15 fusion proteins for 3 days in 96-wellplates at 37 C in a CO₂ incubator. Cells were incubated with 10 μL ofWST-1 for 4 hours before harvesting 150 μL of culture medium for 440 nmabsorbance measurement with a microtiter plate reader. As shown in FIG.19A and FIG. 19B, the c264scTCR/huIL15 fusion protein comprising thewild type IL-15 domain can support the proliferation of CTLL-2 and 32Dβcells at a concentration as low as 40 pM or 1 nM respectively.Interestingly, the fusion protein comprising an IL15 variant with anasparagine to aspartic acid substitution at position 72 with an aminoacid (c264scTCR/huIL15N72D) was much more active that the fusion proteincomprising the wild type IL-15 domain at supporting the proliferation of32Dβ cell line, showing biological activity at a concentration as low as80 pM (FIG. 19B). In this respect the fusion protein comprising IL-15variant (huIL15N72D) showed super agonist activity. In a complex withc264scTCR/IL15RαSushi at one to one ratio, the c264scTCR/huIL15N72D hadsimilar binding ability as c264scTCR/huIL15wt to p53/HLA-A2.1 complex onT2 cells (FIG. 17A) but exhibited increased binding ability toIL-15Rβγ_(c) receptors on 32Dβ cells (FIG. 17C). In contrast, the fusionproteins comprising IL-15 variants with substitutions at position 8(c264scTCR/huIL15D8N or c264scTCR/huIL15D8A), position 65(c264scTCR/huIL15N65A), position 108 (c264scTCR/huIL15Q108A), or adifferent substitution at position 72 (c264scTCR/huIL15N72R) were lessactive in supporting proliferation of both CTLL-2 and 32Dβ cellscompared to c264scTCR/huIL15wt fusion protein (FIG. 19A and FIG. 19B).Similar studies of IL-15-dependent proliferative activity were carriedout with other fusion proteins comprising IL-15 variants and aresummarized in Table 1. The data support the hypothesis that mutations atpositions 8, 61, 65, 72 and 108 of the IL-15 protein can result in IL-15antagonists with decreased binding to IL-15R and little or no activityto stimulate immune responses. The results with the position 72substitutions are unexpected given that one mutant(c264scTCR/huIL15N72R) acted as an IL-15 antagonist whereas a differentmutant (c264scTCR/huIL15N72D) showed increased binding to IL-15 R andenhanced activity at stimulate immune responses.

In a typical circumstance, IL-15 is trans-presented by IL15Rα on adendritic cell surface to IL-15Rβγ_(c) receptors on memory T, NKT, or NKcell to support cell survival and stimulate immune responses. Anantagonist should block the trans-presentation of IL-15 by bindingIL15Rα. To evaluate if the antagonist fusion proteins can compete withc264scTCR/huIL15wt to block its activity to support CTLL-2 cell growth,4×10⁴ CTLL-2 cells were incubated with 0.5 nM of c264scTCR/huIL15wt inthe presence or absence of 50 nM (100-fold molar excess) of variousc264scTCR/huIL15 mutant fusion proteins at 37 C in a CO₂ incubator for24 hours. Cells were incubated with 10 μL of WST-1 for 4 hours beforeharvesting 150 μL of culture medium for 440 nm absorbance measurementwith a microtiter plate reader. As shown in FIG. 19C, the ability ofc264scTCR/huIL15wt to support proliferation of CTLL-2 cells was totallyblocked in the presence of 100-fold more c264scTCR/huIL15D8N,c264scTCR/huIL15D8A, c264scTCR/huIL15D8A/Q108A, c264scTCR/huIL15Q108A,or c264scTCR/huIL15D8N/N65A, and was reduced 62% in the presence ofc264scTCR/huIL15N72R fusion protein. It suggested that these fusionproteins were the antagonists to c264scTCR/IL15 fusion protein. Thisdata indicates that c264scTCR/huIL15 mutant fusion proteins werefunctional antagonist of the IL-15 activity, as expected based on theability of these proteins to bind IL-15Rα but not IL-15Rβγ_(C)receptors.

Similar studies will be carried out with the other TCR/IL15 fusionproteins and IL-15 variants described herein to demonstrate IL-15antagonist and agonist activity. As summarized in Table 1, thesubstitutions at positions 8, 61, 65, 72, and 108 of IL-15 show theability to affect the binding of IL-15 to IL-15R (βγ_(c) chains). Othersubstitutions at positions 92, 101, and 111 of IL-15 will also beassessed as potential binding sites for IL-15R interaction. In addition,combinations of changes including substitutions at all or several ofthese residues may create the effective antagonists or agonists ofIL-15. Including the molecules described above, IL-15 variants to beassessed include those with changes at position 8 to alanine,asparagine, serine, lysine, threonine, or tyrosine; position 61 toalanine, asparagine, serine, lysine, threonine, or tyrosine; position 65to alanine, aspartic acid, or arginine; position 72 to alanine, asparticacid, or arginine; and positions 92, 101, 108, or 111 to alanine orserine.

Example 17 Cell-Cell Conjugation and Immune Cell Retargeting by theTCR/IL15 and TCR/IL15Rα Fusion Proteins and Fusion Protein Complexes

To demonstrate that the fusion proteins or fusion protein complexes canbridge IL-15 receptor-bearing cells with peptide/MHC bearing targetcells, T2 cells will be loaded with either p53 (aa264-272) peptide orcontrol CMVpp65 (aa495-503) peptide and then labeled withdihydroethidium. CTLL-2 cells will be labeled with calcein AM and thetwo labeled cell populations will be mixed and incubated in the presenceor absence of the fusion proteins or fusion protein complexes. In theabsence of the fusion protein complexes or when the T2 cells were loadedwith control peptide, the cells are anticipated to remain as twodistinct populations as assessed by flow cytometry. However, when the T2cells are loaded with p53 (aa264-272) and incubated with the CTLL-2cells in the presence of fusion proteins or complexes, the appearance ofa double staining population of cells would be indicative of conjugationof T2 cells to CTLL-2 cells via the fusion proteins or fusion proteincomplexes.

Similarly, studies can be conducted to demonstrate that the fusionprotein complexes can bridge IL-15 receptor-bearing immune cells withpeptide/MHC bearing target cells and direct immune cytotoxicity againstthe target cells. For example, T2 cells will be loaded with either p53(aa264-272) peptide or control CMVpp65 (aa495-503) peptide and thenlabeled with calcein AM. Immune effector cells bearing IL-15 receptors(i.e. activated NK cells or T cells) will be mixed at different ratiosand incubated under appropriate conditions (i.e. 37 C for 2-4 hours) inthe presence or absence of the fusion protein complex. Cytotoxicity willbe assessed based on release of calcein from the T2 target cells intothe culture media by standard methods. The specific release ofcalcein-AM will be measured or compared to the non-specific control ofspontaneous released calcein-AM. In the absence of the fusion proteincomplex or when the T2 cells were loaded with control peptide, lowlevels of target cell cytotoxicity are expected. However, when the T2cells are loaded with p53 (aa264-272) and incubated with the immuneeffector cells in the presence of fusion protein complex, specific lysisof the T2 cells would be an indication that the immune effector cellsare retargeted against the p53 peptide-presenting cells via the fusionprotein complex. Similar studies will be conducted with tumor cell linespresenting p53 (aa264-272)/HLA-A2.1 complexes as target cells.

Example 18 In vivo Demonstration of Anti-Tumor Effects of IL-15 VariantAgonists, TCR/IL15 Fusion Proteins and Fusion Protein Complexes

To determine if the fusion protein complexes or IL-15 variant agonistshave anti-tumor activity in vivo, an experimental xenograft tumor modelwill be used. Human tumor cell lines expressing p53 (aa264-272)/HLA-A2.1complexes, such as A375 melanoma, MDA-MB-231 mammary adenocarcinoma,PANC1 pancreatic carcinoma, have been employed in similar animalefficacy studies using other TCR-based fusion proteins (5-7). Forexample, A375 human melanoma cells will be injected subcutaneously intothe flank of nude mice and tumors will be allowed to establish for threedays. Tumor bearing mice will be injected intravenously withc264scTCR/huIL15+c264scTCR/huIL15RαSushi complex or an IL-15 variantagonist (dose range—0.1 to 2 mg/kg), or the dose volume equivalent ofPBS daily for four or more days. During the study, tumor size willmeasured and the tumor volumes will be calculated. All mice treated withPBS are expected to develop tumors. Suppression of tumor growth orcomplete tumor regression in some or all the mice treated with thefusion protein complex or IL-15 variant agonist would be an indicationof an antitumor effect of the treatment. Alternative dosing schedulesincluding multi-cycle dosing may also demonstrate the antitumor efficacyof the fusion protein or IL-15 variant agonist. Tumor cell lines lackingp53 (aa264-272)/HLA-A2.1 complexes (such as HT-29 or AsPC-1 (5, 9)) canbe used as controls for antigen specific recognition by thec264scTCR-domain of the fusion protein complex. Alternative fusionprotein complexes comprising other TCR domains (i.e. specific to CMVpp65(aa495-503) peptide (9) could be used as non-tumor targeting controls.

In addition, adoptive cell transfer studies will be carried out inxenograft tumor bearing mice. For example, immune cells bearing theIL-15 receptor, such as naïve or activated (or memory) splenocytes, NKcell or T cells, will be isolated from mice and incubated withc264scTCR/huIL15+c264scTCR/huIL15RαSushi complex or an agonist IL-15variant under conditions permitting binding to the cells. In some casesthe fusion protein complex or an agonist IL-15 variant will be used toactivate the immune cells. The IL-15 variant activated cells or fusionprotein complex-coated cells will then be transferred into nude micebearing A375 tumors. Controls will include transfer of the untreatedimmune cells, the fusion protein complex alone and PBS. Tumor growthwill be monitored and all mice treated with PBS are expected to developtumors. Suppression of tumor growth or complete tumor regression in someor all the mice treated with the IL-15 variant activated cells or fusionprotein complex-coated cells would be an indication of an antitumoreffect of the treatment. Alternatively, the IL-15 variant or fusionprotein complex and immune cells will be administered at the same time,or separately at the same or different times. The immune cells may beautologous or allogeneic in relation with the tumor-bearing host. Thenumber of cells transferred and dosing schedule will be varied to assessand optimize antitumor efficacy. As described above, other tumor linesor fusion protein complexes will be employed to determine the role ofantigen targeting in any observed antitumor activity.

Example 19 In vitro Treatment of Immune Cells with TCR/IL15:TCR/IL15RαFusion Protein Complexes Followed by Adoptive Cell Transfer ProvideImproved Survival in Xenograft Tumor Animal Model

To demonstrate the anti-tumor efficacy of enriched allogenic mouse NKcells preincubated with TCR/IL15:TCR/IL15Rα fusion protein complexes ontumor growth, the following study was carried out using human NSCLCA549A2 tumor cells in an experimental metastasis model in nude mice.

Athymic nude mice (n=4 per group, female, 5-6 week old) wereintravenously (IV) injected through the lateral tail vein with the humanNSCLC tumor cell line A549-A2 at 5×10⁶ cells/mouse. The A549-A2 cellline represents a transfectant of the p53-positive A549 parental linecarrying a vector expressing human HLA-A2.1 cDNA.

Spleens from A2 mice (B6 background) were collected and NK cells wereisolated using a NK cell isolation kit from Miltenyi Biotech, Inc.according to the manufacturer's instruction. Briefly, a single cellsuspension of splenocytes was prepared by homogenizing the spleensthrough a metal screen (60 mesh) in HBSS. Red blood cells were lysed inACK red blood lysing buffer. Cells will be incubated withbiotin-antibody cocktail (10 μL for 10⁷ cells) for 10 min at 4-8 C. Thenumber of leukocytes was determined and 30 μL of buffer (PBS pH 7.2,0.5% BSA and 2 mM EDTA) and 20 μL of anti-biotin MicroBeads per 107cells was added and the mixture was incubated at 4-8 C for 15 min. Thecells were washed in 2 mL buffer and centrifuge at 300×g for 10 min. Thecells were resuspended in 500 μL of buffer for loading to the MACScolumn. The flow through was collected and the purity of the NK cellswas determined using FACScan analysis.

In order to activate the cells, NK cells (5×10⁶) were cultured at 37 Covernight in the presence or absence of c264scTCR/IL15: c264scTCR/IL15Rαfusion protein complex, TCR-IL2 fusion protein or rhIL-2 in T25 flasksin 10 ml RPMI1640 supplemented with 10% FBS. c264scTCR/IL15:c264scTCR/IL15Rα fusion protein complex and TCR-IL2 fusion protein wasadded at a concentration of 0.8 μg/mL and rhIL-2 was added at 0.2 μg/mL.After overnight incubation, cells were harvested and preincubated in 0.5mg/mL c264scTCR/IL15: c264scTCR/IL15Rα fusion protein complex or TCR-IL2fusion protein or 0.125 mg/mL rhIL-2 in 100 μL on ice for 30 min. Afterwash in PBS (1 mL), cells were resuspended in PBS at 10×10⁶ /mL foradoptive transfer.

On day 1, mice were injected i.v. via the tail vein with A549A2 tumorcells (5×10⁶) to establish pulmonary tumors. Fourteen days post tumorcell injection, mice were randomized and divided into 5 groups (n=4).Mice were treated with cyclophosphamide (CTX) via intraperitonealinjection at a dose of 200 mg/kg on days 14 and 21. NK cells(1×10⁶/mouse) preincubated with different fusion proteins or rhIL-2 wereinjected i.v. on days 16 and 22, and mice receiving PBS served ascontrols. A summary of the treatment schedule is a follows:

CTX NK cells Dose Dose Group (mg/kg) Injection (ip) (×10⁶) Injection(iv) CTX 200 Days 14, 21 0 Days 16, 22 CTX + NK/rhIL2 200 Days 14, 21 1Days 16, 22 CTX + NK/MART-1scTCR-IL2 200 Days 14, 21 1 Days 16, 22 CTX +NK/c264scTCR-IL2 200 Days 14, 21 1 Days 16, 22 CTX + NK/c264scTCR/IL15:c264scTCR/ 200 Days 14, 21 1 Days 16, 22 IL15Rα fusion protein complex

Survival of tumor-bearing mice was monitored every day. Mice that becamemoribund were sacrificed and counted as dead. Mice surviving longer than100 days post-tumor injection were considered as cured.

Median survivals for mice in the CTX, CTX+NK/rhIL-2,CTX+NK/MART1scTCR-IL2, CTX+NK/c264scTCR-IL2 and c264scTCR/IL15:c264scTCR/IL15Rα fusion protein complex treatment groups are 52, 67.5,64.5, 85.5, and 80 days, respectively (FIG. 20). Thus, adoptive transferof c264scTCR/IL15: c264scTCR/IL15Rα-activated NK cells resulted a longermedian survival time than observed in tumor-bearing animals treated withchemotherapy alone or with NK cells activated by the non-targetedMARTscTCR-IL2 or rhIL-2. The results from this pilot experiment indicatethat activation and targeting mouse NK cells with c264scTCR/IL15:c264scTCR/IL15Rα may provide enhanced antitumor activity.

Example 20 Enhanced Binding of TCR/IL15:TCR/IL15Rα Fusion ProteinComplexes to IL-15R Bearing Immune Cells as Evidenced by an ExtendedCell Surface Residency Time

The cell surface residency time of the fusion protein complexes on theIL-15R-bearing cells may influence the ability of the fusion protein totarget or bridge effector cells with the TCR-specific tumor cells. Toinvestigate this, binding of the scTCR/IL-15 fusion proteins,TCR/IL15:TCR/IL15Rα fusion protein complexes and recombinant IL-15 toIL-15RαβγC receptor-bearing CTLL-2 cells and IL-15RβγC receptor-bearingCTLL-2 cells 32Dβ cells will be directly compared by flow cytometry.Following incubation with the various proteins, cells will be washed andincubated in media at 37° C. for up to 180 min and the level of proteinsremaining on the cell surface was detected with PE-labeled anti-IL-15mAb. Comparisons between the initial time zero staining and subsequenttimes will allow determination of the cell surface residency time foreach proteins binding to IL-15R. Increased cell surface residency timeof the scTCR/IL-15 fusion proteins or TCR/IL15:TCR/IL15Rα fusion proteincomplexes compared to IL-15 would be an indication of enhanced and morestable receptor binding activity.

Example 21 Increased in vivo Half Life of TCR/IL15 Fusion Proteins andTCR/IL15:TCR/IL15Rα Fusion Protein Complexes Compared to IL-15 in Mice

The pharmacokinetic parameters of c264scTCR/IL-15, c264scTCR/IL15:c264scTCR/IL15Rα complex, recombinant IL-15 or soluble IL-15:IL-15Racomplex will be evaluated in the HLA-A2.1/Kb-transgenic mouse strain.The presence of the HLA-A2.1 domain, for which c264scTCR/IL-2 isrestricted, may influence the pharmacokinetics of this fusion proteinand should give a more relevant “humanized” view of the pharmacokineticsthan other mouse strains. Mice will be injected intravenous with molarequivalent amounts of c264scTCR/IL-15, c264scTCR/IL15: c264scTCR/IL15Rαcomplex, recombinant IL-15 or soluble IL-15:IL-15Ra complex and bloodwill be collected at various time points from 5 minutes to two weekspost injection. Serum concentrations of the fusion proteins will beevaluated using ELISA formats, disclosed above. Concentrations of IL-15were detected with a standard IL-15-specific ELISA.

The in vivo pharmacokinetic parameters of c264scTCR/IL-15,c264scTCR/IL15: c264scTCR/IL15Rα complex, recombinant IL-15 or solubleIL-15:IL-15Ra complex will be determined using curve fitting software(e.g., WinNonlin). Elevated Cmax values, increased serum half life ordecreased clearance for the c264scTCR/IL-15 or c264scTCR/IL15:c264scTCR/IL15Rα complex compared to recombinant IL-15 or solubleIL-15:IL-15Ra complex would be an indication that generation of theTCR-IL15 fusion or TCR/IL15:TCR/IL-15Ra complex provides more favorablepharmacokinetics that is observed with IL-15 alone.

Example 22 In vivo Demonstration of Immunosuppressive Effects of IL-15Variant Antagonists, TCR/IL15 Fusion Proteins and Fusion ProteinComplexes

To determine if the fusion protein complexes or IL-15 variantantagonists have immuno-suppressive activity in vivo, an experimentalautoimmune arthritis model will be used. It has been demonstrated thatautoimmune arthritis can be induced following administration of type IIcollagen (CII) in HLA-DR4-transgenic mice (Rosloniec et al. 1998. JImmunol. 160:2573-8). Additionally, CII-specific T-cells involved in thepathology of this disease have been characterized. The TCR genes fromthese T-cells will be used to construct the appropriate expressionvectors and host cell lines to generate CIIscTCR/IL15 comprising IL-15variant antagonists and CIIscTCR/IL15RαSushi fusion proteins asdescribed in previous examples. Following induction of arthritis by CIIadministration, the HLA-DR4-transgenic mice will be injectedintravenously with CIIscTCR/IL15-antagonst+CIIscTCR/IL15RαSushi complexor an IL-15 variant antagonist (dose range—0.1 to 2 mg/kg), or the dosevolume equivalent of PBS daily for four or more days. During the study,the mouse paw joints will be evaluated and scored for the degree ofinflammation on a scale of 0 to 4. All mice treated with PBS areexpected to develop arthritis. Suppression of arthritis (e.g. decreasedincidence or clinical score) in some or all the mice treated with thefusion protein complex or IL-15 variant would be an indication of animmunosuppressive effect of the treatment. Alternative dosing schedulesincluding multi-cycle dosing may also demonstrate the immunosuppressiveefficacy of the fusion protein or IL-15 variant. Alternative fusionprotein complexes comprising other TCR domains (i.e. specific to p53peptide) could be used as non-disease targeting controls to demonstratespecificity of the targeted TCR fusion proteins ability to directimmunosuppressive activity to the disease site.

Incorporation by Reference

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

References

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What is claimed is:
 1. An isolated soluble fusion protein complexcomprising at least two soluble fusion proteins, wherein the firstfusion protein comprises (a) a first single chain T-cell receptor(scTCR) covalently linked to (b) interleukin-15 (IL-15) polypeptide; andthe second fusion protein comprises (c) a second scTCR covalently linkedto (d) soluble interleukin-15 receptor alpha (IL-15Ra) polypeptide;wherein the IL-15 polypeptide of the first fusion protein binds to thesoluble IL-15Ra polypeptide or of the second fusion protein to form asoluble fusion protein complex, and further wherein the first scTCR andthe second scTCR comprise a TCR V-α chain covalently linked to a TCR V-βchain by a peptide linker sequence.
 2. The soluble fusion proteincomplex of claim 1 wherein the first scTCR and the second scTCR areidentical, thereby creating a multivalent TCR fusion protein complexwith increased binding activity.
 3. The soluble fusion protein complexof claim 1 wherein the first scTCR is specific for recognition of aparticular antigen.
 4. The soluble fusion protein complex of claim 3wherein the antigen for the first scTCR domain comprises peptide antigenpresented in an MHC or HLA molecule.
 5. The soluble fusion proteincomplex of claim 1, wherein the first scTCR or second scTCR furthercomprises a soluble TCR Cβ chain fragment covalently linked to a TCR V-βchain.
 6. The soluble fusion protein complex of claim 1, wherein thefirst scTCR or second scTCR further comprises a soluble TCR Cα chainfragment covalently linked to a TCR V-α chain.
 7. The soluble fusionprotein complex of claim 1, wherein the IL-15 polypeptide is an IL-15variant comprising a different amino acid sequence than native IL-15polypeptide.
 8. The soluble fusion protein complex of claim 7, whereinthe IL-15 variant functions as an IL-15 agonist or antagonist.
 9. Thesoluble fusion protein complex of claim 7, wherein the IL-15 variant hasincreased or decreased binding activity for the IL-15RβγC receptorscompared to the native IL-15 polypeptide.
 10. The soluble fusion proteincomplex of any one of claims 7-9, wherein the sequence of the IL-15variant has at least one amino acid change compared to the native IL-15sequence.
 11. The soluble fusion protein complex of claim 10, where theamino acid change is an amino acid substitution or deletion in thedomain of IL-15 that interacts with IL-15Rβ and/or γC.
 12. The solublefusion protein complex of claim 10, where the amino acid change is oneor more amino acid substitutions or deletions at position 8, 61, 65, 72,92, 101, 108, or 111 of the mature human IL-15 sequence (SEQ ID NO:1).13. The soluble fusion protein complex of claim 10, wherein the aminoacid change is the substitution of D to N or A at position 8, D to A atposition 61, N to A at position 65, N to R at position 72 or Q to A atposition 108 of the mature human IL-15 sequence, or any combination ofthese substitutions.
 14. The soluble fusion protein complex of claim 13,wherein the amino acid change results in an IL-15 variant that has IL-15antagonist activity or decreased binding activity for the IL-15RβγCreceptors compared to the native IL-15 polypeptide.
 15. The solublefusion protein complex of claim 10, wherein the amino acid change is thesubstitution of N to D at position 72 of the mature human IL-15sequence.
 16. The soluble fusion protein complex of claim 15, whereinthe amino acid change results in an IL-15 variant that has IL-15 agonistactivity or increased binding activity for the IL-15RβγC receptorscompared to the native IL-15 polypeptide.
 17. The soluble fusion proteincomplex of claim 1 wherein the first scTCR is covalently linked to IL-15polypeptide, by a polypeptide linker sequence.
 18. The soluble fusionprotein complex of claim 1 wherein the second scTCR is covalently linkedto IL-15Ra polypeptide, by a polypeptide linker sequence.
 19. Thesoluble fusion protein complex of claim 1 wherein the IL-15Rapolypeptide comprises the extracellular domain of the IL-15 receptoralpha capable for binding IL-15 polypeptide.
 20. The soluble fusionprotein complex of claim 1 wherein the IL-15Ra polypeptide compriseeither the IL-15a sushi domain or the IL-15aΔE3 domain.
 21. A nucleicacid sequence encoding the first fusion protein of claim
 1. 22. A DNAvector comprising the nucleic acid sequence of claim
 21. 23. A nucleicacid sequence encoding the second fusion protein of claim
 1. 24. A DNAvector comprising the nucleic acid sequence of claim
 23. 25. A DNAvector comprising nucleic acid sequences of claims 21 and
 23. 26. Amethod for making a soluble fusion protein complex of claim 1, themethod comprising: a) introducing into a first host cell a DNA vectorencoding the first fusion protein, b) culturing the first host cell inmedia under conditions sufficient to express the first fusion protein inthe cell or the media; c) purifying the first fusion protein from thehost cells or media, d) introducing into a second host cell a DNA vectorencoding the second fusion protein, e) culturing the second host cell inmedia under conditions sufficient to express the second fusion proteinin the cell or the media; and f) purifying the second fusion proteinfrom the host cells or media, and g) mixing the first and second fusionprotein under conditions sufficient to allow binding between IL-15domain of a first fusion protein and the soluble IL-15Ra domain of asecond fusion protein to form the soluble fusion protein complex.